Methods of treating cancer

ABSTRACT

Provided herein are, inter alia, methods of treating cancer by administering to a subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist or a combination of an adenosine-A2A(A2A) receptor antagonist and a programmed cell death protein 1(PD-1) signaling pathway inhibitor. Further provided are pharmaceutical compositions including an A2A receptor antagonist, a PD-1 signaling pathway inhibitor and a pharmaceutically acceptable excipient. Further provided are methods of detecting cellular effects, for example expression of pCREB, before, after or during adenosine receptor antagonist treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application filed under 35 U.S.C. §371, of International Patent Application No. PCT/US2016/068459, filedDec. 22, 2016, which claims priority to U.S. Provisional Application62/387,383, filed Dec. 24, 2015, U.S. Provisional Application No.62/324,211, filed Apr. 8, 2016, U.S. Provisional Application No.62/350,602, filed Jun. 15, 2016, U.S. Provisional Application No.62/421,109, filed Nov. 11, 2016, and U.S. Provisional Application No.62/421,171, filed Nov. 11, 2016, which are hereby incorporated byreference in their entirety and for all purposes.

BACKGROUND OF THE INVENTION

The goal of immunotherapy is to drive cytotoxic T-cell responses toeradicate cancer. To prevent reaction to self-antigens, or overreaction,multiple inhibitory checkpoint signals exist including PD½, CTLA4 andadenosine. Extracellular adenosine, a purine nucleoside, is producedduring acute, inflammatory processes by conversion from adenosinetriphosphate (ATP) through ectonucleotidases CD73 and CD39 expressed onthe cell surface of multiple tissue types. Adenosine is normallyupregulated to protect a host from over-injury in response to suchstimuli as infection or ischemia by binding its extracellular, G-proteincoupled receptors on target cells (including MR, A2AR, A2BR, and A3R)and begin healing {Hasko 2008}. However, multiple tumor types canactively sustain extracellular adenosine levels well beyond acute phasereactions to dampen a host's immune response through multiple mechanisms{Antionioli 2013}. Increases in adenosine in the microenvironment bymalignant cells recruits regulatory T-cells (Treg), which expresssubstantial CD39, to the area and further drive up adenosine levels{Sica 2010}.

Cancer cells also appear to directly utilize adenosine. As a result,adenosine causes inefficient presentation of tumor antigens to theadaptive system and enhances tumor growth. Thus, there is a need in theart for effective cancer treatments. The methods and compositionsprovided herein address these and other deficiencies in the art.

BRIEF SUMMARY OF THE INVENTION

In one aspect, a method of treating cancer in a subject in need thereofis provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist and a programmed cell death protein 1 (PD-1) signalingpathway inhibitor.

In another aspect, a method of treating cancer in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. Thesymbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Thesymbols m₁, m₂ and m₃ are independently an integer from 1 to 2. And thesymbols v₁, v₂ and v₃ are independently an integer from 1 to 2.

In one aspect, a method of activating a T cell is provided. The methodincludes contacting the T cell with an A2A receptor antagonist, whereinthe A2A receptor antagonist is a compound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. Thesymbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Thesymbols m₁, m₂ and m₃ are independently an integer from 1 to 2. Thesymbols v₁, v₂ and v₃ are independently an integer from 1 to 2.

In one aspect, a method of inhibiting A2A receptor activity of a cell isprovided. The method includes contacting the cell with an A2A receptorantagonist, wherein the A2A receptor antagonist is a compound offormula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. Thesymbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Thesymbols m₁, m₂ and m₃ are independently an integer from 1 to 2. Thesymbols v₁, v₂ and v₃ are independently an integer from 1 to 2.

In one aspect, a method of increasing an anti-tumor immune response in asubject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist and a programmed cell death protein 1 (PD-1)signaling pathway inhibitor.

In another aspect, a method of increasing an anti-tumor immune responsein a subject in need thereof is provided. The method includesadministering to the subject a therapeutically effective amount of anadenosine-A2A (A2A) receptor antagonist, wherein the A2A receptorantagonist is a compound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. Thesymbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Thesymbols m₁, m₂ and m₃ are independently an integer from 1 to 2. Thesymbols v₁, v₂ and v₃ are independently an integer from 1 to 2.

In one aspect, a method of increasing the amount of CD8-positive cellsrelative to the amount of regulatory T cells in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist and a programmed cell death protein 1 (PD-1) signalingpathway inhibitor.

In one aspect, a method of increasing the amount of CD8-positive cellsrelative to the amount of regulatory T cells in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein the A2A receptor antagonist is a compound offormula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. Thesymbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Thesymbols m₁, m₂ and m₃ are independently an integer from 1 to 2. Thesymbols v₁, v₂ and v₃ are independently an integer from 1 to 2.

In one aspect, a method of decreasing tumor volume in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist and a programmed cell death protein 1 (PD-1) signalingpathway inhibitor.

In one aspect, a method of decreasing tumor volume in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein the A2A receptor antagonist is a compound offormula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. Thesymbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Thesymbols m₁, m₂ and m₃ are independently an integer from 1 to 2. Thesymbols v₁, v₂ and v₃ are independently an integer from 1 to 2.

In one aspect, a method of enhancing anti-tumor immune memory in asubject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist and a programmed cell death protein 1 (PD-1)signaling pathway inhibitor.

In one aspect, a method of enhancing anti-tumor immune memory in asubject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist, wherein the A2A receptor antagonist is acompound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. Thesymbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Thesymbols m₁, m₂ and m₃ are independently an integer from 1 to 2. And thesymbols v₁, v₂ and v₃ are independently an integer from 1 to 2.

In one aspect, a method of increasing global immune activation in asubject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist, wherein the A2A receptor antagonist is acompound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. Thesymbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Thesymbols m₁, m₂ and m₃ are independently an integer from 1 to 2. And thesymbols v₁, v₂ and v₃ are independently an integer from 1 to 2.

In one aspect, a method of treating cancer in a subject in need thereofis provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist of formula:

wherein the adenosine-A2A (A2A) receptor antagonist is administered at100 mg twice a day (BID).

In one aspect, a method of treating cancer in a subject in need thereofis provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist of formula:

and a therapeutically effective amount of atezolizumab.

In one aspect, a pharmaceutical composition including an A2A receptorantagonist, a PD-1 signaling pathway inhibitor and a pharmaceuticallyacceptable excipient is provided.

In one aspect, a pharmaceutical composition including an A2A receptorantagonist of formula:

and a pharmaceutically acceptable excipient is provided, wherein theadenosine-A2A (A2A) receptor antagonist is present at 100 mg.

In one aspect, a pharmaceutical composition including an adenosine-A2A(A2A) receptor antagonist of formula:

atezolizumab and a pharmaceutically acceptable excipient is provided.

In one aspect, a method of activating a T cell is provided. The methodincludes contacting the T cell with an A2A receptor antagonist, whereinthe adenosine-A2A (A2A) receptor antagonist is a compound of formula:

In one aspect, a method of inhibiting A2A receptor activity of a cell isprovided. The method includes contacting said cell with an A2A receptorantagonist, wherein the A2A receptor antagonist is a compound offormula:

In one aspect, a method of increasing an anti-tumor immune response in asubject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist, wherein the adenosine-A2A (A2A) receptorantagonist is a compound of formula:

In one aspect, a method of increasing the amount of CD8-positive cellsrelative to the amount of regulatory T cells in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein the adenosine-A2A (A2A) receptor antagonist is acompound of formula:

In one aspect, a method of decreasing tumor volume in a subject in needthereof is provided. The method includes administering to said subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein the adenosine-A2A (A2A) receptor antagonist is acompound of formula:

In one aspect, a method of enhancing anti-tumor immune memory in asubject in need thereof is provided. The method includes administeringto said subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist, wherein the adenosine-A2A (A2A) receptorantagonist is a compound of formula:

In one aspect, a method of detecting a phosphorylated cAMP responseelement-binding protein (pCREB) in a B-cell or T-cell of a mammaliansubject is provided. The method includes:

-   -   (i) obtaining a blood sample from a mammalian subject;    -   (ii) contacting the blood sample with an adenosine receptor        agonist;    -   (iii) contacting the blood sample with a pCREB detection agent        and a blood cell detection agent, wherein the blood cell        detection agent includes a B-cell detection agent or T-cell        detection agent, thereby forming a T-cell-detection agent        complex or a B-cell-detection agent complex; and    -   (iv) detecting the T-cell detection agent complex or the B-cell        detection complex thereby detecting the pCREB in a T-cell or        B-cell.

In one aspect, the adenosine receptor agonist includes adenosine,5′-N-Ethylcarboxamidoadenosine (NECA), or an analog thereof. In oneaspect, the pCREB detection agent includes an antibody against pCREB. Inone aspect, the B cell detection agent includes an antibody against CD19and/or an antibody against CD20. In one aspect, the T cell detectionagent includes an antibody against CD3, CD4 and/or an antibody againstCD8.

In one aspect, the method further includes contacting the blood samplewith a fixation agent and cell permeabilizing agent after contacting theblood sample with an adenonsine receptor agonist and prior to contactingthe blood sample with a pCREB detection agent. In one aspect, the methodfurther includes contacting the blood sample with an apoptotic celldetection agent. In one aspect, the apoptotic cell detection agentincludes an antibody against cPARP. In one aspect, the method furtherincludes, prior to obtaining the blood sample, administering to themammalian subject an adenosine receptor antagonist.

In one aspect, the adenosine receptor antagonist includes an A2areceptor antagonist or an A2b receptor antagonist. In one aspect, themethod further includes, prior to obtaining the blood sample,administering to the mammalian subject an anti-cancer agent. In oneaspect, the anti-cancer agent includes a PD-L1 antagonist. In oneaspect, the PD-L1 antagonist includes atezolizumab. In one aspect, themethod further includes, contacting the blood sample with a cell subsetdetection agent. In one aspect, the cell subset detection agent includesa naïve cell detection agent, a memory cell detection agent, or aneffector cell detection agent. In one aspect, the cell subset detectionagent includes an antibody against CD27 or an antibody against CD45RA.In one aspect, the blood sample is collected from circulating blood. Inone aspect, the blood sample includes an intratumoral sample.

In one aspect, a method of treating a subject with cancer is provided.The method includes:

-   -   (i) obtaining a blood sample from a subject with cancer;    -   (ii) detecting a level of pCREB induced by an adenosine receptor        agonist in the sample;    -   (iii) administering an effective amount of an adenosine receptor        antagonist to the subject.

In one aspect, the detecting of the level of pCREB induced in the sampleincludes:

-   -   (a) contacting the blood sample with an adenosine receptor        agonist; and    -   (b) contacting the blood sample with a pCREB detection agent and        a blood cell detection agent, wherein the blood cell detection        agent includes a B-cell detection agent or T-cell detection        agent.

In one aspect, the pCREB detection agent includes an antibody againstpCREB. In one aspect, the B cell detection agent includes an antibodyagainst CD19 and/or against CD20. In one aspect, the T cell detectionagent includes an antibody against CD3, CD4 and/or an antibody againstCD8. In one aspect, detecting the level of pCREB induced in the subjectcomprises measuring a level of pCREB in B cells or T cells prior to theadministering of the effective amount of an adenosine receptorantagonist to the subject.

In one aspect, the method further includes: (iv) detecting a level ofpCREB induced in said sample following said administering of theeffective amount of adenosine receptor antagonist to said subject. Inone aspect, the detecting of the level of pCREB induce in said samplecomprises measuring a level of pCREB induced in B cells or T cellsfollowing said administering of the effective amount of adenosinereceptor antagonist to said subject.

In one aspect, the method includes increasing a dose of an adenosinereceptor antagonist based on the level of pCREB induced in said B cellsor T cells.

In one aspect a permeabilized blood cell is provided. The permeabilizedblood cell includes a pCREB detection agent and a blood cell detectionagent, wherein the blood cell detection agent includes a B-celldetection agent or T-cell detection agent and the permeabilized bloodcell includes a permeabilized B-cell or permeabilized T-cell. In oneaspect, the permeabilized blood cell further includes an apoptotic celldetection agent. In one aspect, the apoptotic cell detection agentincludes an antibody against cPARP. In one aspect the permeabilizedblood cell further includes a mature cell detection agent. In oneaspect, the mature cell detection agent includes antibody against CD27or an antibody against CD45RA.

In one aspect, a container including an adenosine receptor agonist incombination with the permeabilized cell as described above is provided.

In one aspect, a flow cytometer including the permeabilized blood cellas described above is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: CPI-444±Anti-PD-1 In Early CT26 Model. Therapeutic synergy incombination with anti-PD-1.

FIG. 2: CPI-444±Anti-PD-L1 In MC38 Model. Combination treatment inhibitstumor growth.

FIG. 3: Efficacy Model: MC38 Colon Cancer. Synergy in combination withanti-PD-L1.

FIG. 4: Efficacy Model: MC38 Colon Cancer. Skewing toward anti-tumorimmune response in tumors.

FIG. 5: Efficacy Model: MC38 Colon Cancer. Skewing toward anti-tumorimmune response in tumors.

FIG. 6: CPI-444 Inhibits cAMP Production in T cells

FIG. 7: CPI-444 Restores IL-2 and IFNγ From Activated T Cells. Primaryhuman PBMCs were cultured for 1 hour in the presence of an A2Ar agonist(NECA or CGS21680, 1 μm) to stimulate the effects of adenosine on immunecell function. Purified anti-CD3 and anti-CD28 monoclonal antibodies (1ug/ml) were then added to activate T cells for 48 hours. NECA andCGS21680 suppressed release of the Th1 cytokines IL-1 and IFNγ,mimicking the immunosuppressive effects of adenosine signaling. Blockadeof A2AR with CPI-444 (1 μM) neutralized the immunosuppressive effects ofNECA and CGS21680 and restored IL-2 and IFNγ secretion back to levelsobserved in the absence of exogenous adenosine signaling (DMSO control).

FIG. 8: CPI-444 Does Not Affect Tumor Cell Proliferation In Vitro

FIG. 9: CPI-444 Restores pERK In CD4⁺ T Cells

FIG. 10: CPI-444 Prevents pCREB Induction in B Cells

FIG. 11: CPI-444 Inhibits EL4 Tumor Growth In Regional Lymph Nodes

FIG. 12: CPI-444 Inhibits Growth of MC38 Tumors

FIG. 13: CT26 Model: Combo extends long-term survival in 80% mice. Oraladministration of control vehicle (40% solution ofhydroxypropyl-beta-cyclodextrin) or CPI-444 (100 mg/kg) was initiatedthe same day tumors were engrafted (Day 0). Treatment continued for 12days. Half of the mice in the vehicle control group as well as half themice in the CPI-444 treatment group received anti-PD-1 mAb (RMP1-14, 100ug/mouse, i.p.) on days 7, 9, 11, and 13. Administration of anti-PD-1 orCPI-444 resulted in an inhibition of tumor growth, however, tumors werenot completely eradicated by either treatment. Administration of CPI-444in combination with anti-PD-1 stabilized or eliminated tumors in 8/9mice, resulting in improved overall survival for more than 3 weeksfollowing the last dose of CPI-44 or anti PD-1 antibody.

FIGS. 14A and 14B: MC38 Model: CPI-444 eliminates tumors in 30% of mice.Combo eliminates tumors in 50% of mice. MC 38 mouse colon cancer cellswere engrafted onto the back of syngeneic C57Bl/6 mice. Oraladministration of control vehicle or CPI-444 (100 mg/kg) was initiatedthe same day tumors were engrafted (Day 0). Treatment continued for 12days. Half of the mice in the vehicle control group as well as half themice in the CPI-444 treatment group received anti-PD-L1 mAb (10F.9G2,200 ug/mouse, i.p.) on days 7, 10, 13, and 16. Administration ofanti-PD-L1 or CPI-444 resulted in an inhibition of tumor growth,however, tumors were not completely eradicated by either treatment. Incontrast, administration of CPI-444 in combination with anti-PD-L1stabilized or eliminated tumors in 5/10 mice.

FIGS. 15A and 15B: MC38 Model: CPI-444 eliminated tumors in 30% of mice.100% protected from rechallenge. Nine mice that achieved complete tumorgrowth inhibition at the end of the CPI-444 dose response study (FIG.15A) were monitored for signs of reoccurrence for an additional 6 weeks.No tumor growth was observed, indicating that the tumor had been fullyeliminated. These mice were challenged with the new engraftment of MC38tumor cells. Modest tumor growth was observed in the first 5 days afterrechallenge, however the tumors were fully rejected in all 9 mice overthe following 15 days (FIG. 15B). Notably, tumor elimination occurred inthe absence of any additional CPI-444 treatment. These results clearlydemonstrate that CPI-444 treatment can elicit durable systemicanti-tumor immune memory.

FIGS. 16A and 16B: MC38 Model: Established tumors eliminated in 8/9mice. FIG. 16A: MC38 colon cancer cells were engrafted onto the back ofsyngeneic C57BL/6 mice. Mice were treated with CPI-444 (100 mg/kg),anti-PD-L1 (10F.9G2, 200 ug), or appropriate controls as indicated. Alltreatment regimens resulted in an inhibition of tumor growth, howevertherapeutic efficacy was optimal when CPI-444 was administered prior toor concurrent with anti-PD-L1 (FIG. 16B). The combination of CPI-444with anti PD-L1 was particularly effective when initiated on establishedtumors (Day 7) and resulted in full tumor elimination in 8/9 mice.

FIG. 17: MC38 Colon Cancer: Cartoon of Dosing Strategies: Determineoptimal order of CPI-444 and anti-PD-L1.

FIGS. 18A and 18B: MC38 Colon Cancer: All treatments started on Day 7(established tumors). Size of tumor volume (FIG. 18A). Cartoon of dosingstrategies (FIG. 18B).

FIGS. 19A and 19B: T Cell activation in treated subjects. Whole bloodwas collected on Day 1 of Cycles 1, 2, 4 & 8 for flow analysis. Thepercentage of CD4 and CD8 T cells that stained PD-1+(FIG. 19A) orCD45RA− (memory/effector cells) (FIG. 19B) is shown. Each linerepresents a single subject across time. CPI-444 treatment increasesPD-1+/CD8+ and memory cell frequencies as a single agent and incombination cohorts, suggesting activation of T cell mediated immunity.

FIGS. 20A-20C: CPI-444 is associated with changes in T cell repertoire.Whole blood was collected on Day 1 of Cycles 1 and 2 and PBMCs wereprepared. DNA was extracted from PBMCs and sequenced for TCRβ repertoireby Adaptive Biotechnologies. Expansion of pre-existing and new T cellclones is observed in response to treatment with single agent CPI-444(FIG. 20A). Morisita Index measures T cell repertoir similaritycomparing pre- and post-dose PBMCs. A Morisita Index of 1 is equal toidentity, indicating no longitudinal change. Morisita Index distributionin single agent and combination cohorts (FIG. 20B). Graph showingMorisita Index by cohort (FIG. 20C). CPI-444 induces robust changes inTCR repertoire in some patients treated with single agent CPI-444 and incombination with Tecentriq®. Changes were driven predominantly by TCRclone expansion (clonality).

FIGS. 21A-21C: Efficacy by PD-L1 status and prior anti-PD-1 treatmentstatus. The disease is stable in anti-PD-L1 naïve and refractory as wellas patient PD-L1+ and PD-L1-tumors (FIG. 21A and FIG. 21B). Tumorregression in a Nivolumab-refractory lung cancer patient (Single agentCPI-444 100 mg po BID×28 days/cycle)(FIG. 21C). In FIG. 21C, Patient'sMorisita Index was 0.84 and increased clonality was observed followingtreatment.

FIGS. 22A-22C: Relationship between TCR repertoire and efficacy. TCRrepertoire changes are similar between patients that are naïve andrefractory to prior anti-PD-1 therapy and may associate with efficacy(FIG. 22A). FIG. 22B shows the change in tumor size relative to baselineplotted against the Morisita Index. FIG. 22C shows the change in tumorsize relative to baseline plotted against baseline clonality.

FIGS. 23A-23C: CPI-444 efficacy requires CD8+ T cells. MC38 mouse coloncancer cells were engrafted onto the back of syngeneic C57BL/6 mice.Oral administration of control vehicle or CPI-444 (100 mg/kg) wasinitiated 7 days after tumors were engrafted (Day 0) (FIG. 23C).Treatment continued for more than 9 days (FIG. 23C). Half of the mice inthe vehicle control group as well as half the mice in the CPI-444treatment group received anti-PD-L1 mAb (10F.9G2, 200 ug/mouse, i.p.) ondays 7, 10, 13, and 16 (FIG. 23C). 100 ug of Anti-mCD4 (Clone GK1.5)and/or 500 ug of Anti-mCD8 (Clone 53-6.72) was administered on day 6. Tcell depletion was verified by flow analysis. FIGS. 23A and 23B show thetumor volume at different time points since engraftment for the dosingcohorts. These results suggest CD8+ T cells are required for theefficacy of CPI-444 alone or in combination with Anti-PD-L1.

FIG. 24: A schematic showing the role of CPI-444 in CREBphosphorylation.

FIG. 25: A Graph indicating that 5′N-ethylcarboxamido-adenosine (NECA),NECA, a synthetic adenosine analog, activates CREB in whole blood.

FIG. 26: A schematic showing a pharmacokinetic time course of pCREBinduction in CPI-444 dosing. The pCREB assay is performed at: Day 1before dosing; Day 14 with PK time course. Concentrations used are: 50mg BID, 100 mg BID, and 200 mg QD.

FIG. 27: A series of graphs charting pCREB induction in B cells in asubject receiving 200QD CPI-444 for 14 days. Trough refers topharmacokinetic troughs as seen in FIG. 26.

FIG. 28: A series of graphs charting pCREB induction in B cells in asubject receiving 50 mg BID CPI-444+Atezolizumab for 14 days. Troughrefers to pharmacokinetic troughs as seen in FIG. 26.

FIG. 29: A graph showing CREB phosphorylation in B cells at differentconcentrations of NECA prior to adenosine receptor antagonist treatmentand after 14 days of treatment at trough, 1.5 hour, 3 hour, 5 hour and 8hour time points for a subject receiving CPI-444 alone and a subjectreceiving combination therapy of CPI-444 and atezolizumab.′

FIG. 30: A graph showing inhibition of CREB phosphorylation in B cellsrelative to baseline signaling at different concentrations of NECA after14 days of adenosine receptor antagonist treatment at trough, 1.5 hour,3 hour, 5 hour and 8 hour time points for subjects receiving CPI-444alone (subject 100301: 200 mg QD CPI-444; and subject 100303: 100 mg BIDCPI-444) and subjects receiving combination therapy of CPI-444 andatezolizumab (subject 100302: 50 mg BID CPI-444+atezo; and subject100402: 50 mg BID CPI-444+atezo).

FIG. 31: A graph showing CREB phosphorylation in T cells at differentconcentrations of NECA at trough, 1.5 hour, 3 hour, 5 hour and 8 hourtime points for a subject receiving CPI-444 alone (200QD CPI-444 for 14days) and a subject receiving combination therapy of CPI-444 andatezolizumab (50BID CPI-444+atezolizumab for 14 days).

FIG. 32: Phase 1/1B clinical trial design. Step 1 shows the biomarkerobjectives to 1) inform dose selection and schedule usingpharamacodynamics assays (pCREB and immune activation markers) and 2)explore relationships between efficacy and biomarkers, e.g., immuneactivation in serial peripheral blood and tumor biopsy samples. Step 2shows the trial design.

FIGS. 33A-33B: CPI-444 blocks A2AR in treated subjects. Whole blood wascollected on Day 1 pre-treatment and on Day 14 at pre-dose and post doseat 1.5 hr, 3 hr, 5.5 hr and 8 hr time points. Blood was activated withan adenosine analog (NECA) and subsequently stained for intracellularphospho-CREB (pCREB) and cell lineage markers for flow cytometry. Foreach Day 14 time point, the percent inhibition of NECA-induced pCREB isrelative to baseline. FIG. 33A is a graph showing the inhibition ofpCREB relative to baseline levels over time. Complete and sustainedinhibition was seen in 7 of 7 patients treated with 100 mg BID singleagent CPI-444. Variable inhibition was seen in 200 mg QD and 50 mg BIDcohorts. FIG. 33B shows the inhibition of pCREB relative to baseline fordifferent concentrations of plasma CPI-444. PK/PD analysis supports 100mg BID as the optimal dose for Step 2 dose expansion cohorts. The A2ARpathway is completely inhibited at CPI-444 exposures greater than 2000ng/mL.

FIGS. 34A-34D: Graphs showing pCREB percent inhibition in B cells acrossthe 8 hr time course of Day 14. Each line represents a single patientand each graph represents a different does used in step 1 of theclinical trial. FIG. 34A shows pCREB percent inhibition in B cells inpatients who received 50 mg BID of CPI-444. FIG. 34B shows pCREB percentinhibition in B cells in patients who received 100 mg BID of CPI-444.FIG. 34C shows pCREB percent inhibition in B cells in patients whoreceived 200 QD of CPI-444. The majority of patients in the 100 mg BIDcohort demonstrate high pCREB inhibition at trough and near completeinhibition after taking their morning dose. FIG. 34D is a dot plotshowing the change in percent inhibition between trough (0 hr) and peak(3 hr). There is little fluctuation from trough to peak in the 100 mgBID dosing group, making 100 mg BID an appropriate dose for continuousfunctional inhibition. The 50 mg BID is not high enough for sustainedinhibition and the 200 mg QD dose achieves high peak levels but is notmaintained at trough since CPI-444 is administered once per day.

FIGS. 35A-35B: Percent inhibition of CREB phosphorylation by plasmalevels of CPI-444. Each dot in FIG. 35A and FIG. 35B represents a singletime point from a single subject. For plasma levels greater than 2,000ng/mL near complete inhibition was observed in B cells (FIG. 35A) andCD4+ T cells (FIG. 35B).

FIG. 36: pCREB inhibition is correlated between B cells and CD4+ Tcells. Each dot represents a single time point for a single subject. Thex-axis shows pCREB percent inhibition in B cells and the y-axis showspCREB percent inhibition in CD4+ Tcells. There is a strong correlationbetween inhibition in B cells and CD4+ T cells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchednon-cyclic carbon chain (or carbon), or combination thereof, which maybe fully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkylgroup is one having one or more double bonds or triple bonds. Examplesof unsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. An alkoxy is an alkyl attached to theremainder of the molecule via an oxygen linker (—O—). An alkyl moietymay be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. Analkyl moiety may be fully saturated. An alkenyl may include more thanone double bond and/or one or more triple bonds in addition to the oneor more double bonds. An alkynyl may include more than one triple bondand/or one or more double bonds in addition to the one or more triplebonds.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms. Theterm “alkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched non-cyclicchain, or combinations thereof, including at least one carbon atom andat least one heteroatom (e.g. O, N, P, Si, and S), and wherein thenitrogen and sulfur atoms may optionally be oxidized, and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) (e.g. O, N,P, S, and Si) may be placed at any interior position of the heteroalkylgroup or at the position at which the alkyl group is attached to theremainder of the molecule. Examples include, but are not limited to:—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and —CN. Up totwo or three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. A heteroalkyl moiety may include oneheteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includetwo optionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include three optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include fouroptionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include five optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8optionally different heteroatoms (e.g., O, N, S, Si, or P). The term“heteroalkenyl,” by itself or in combination with another term, means,unless otherwise stated, a heteroalkyl including at least one doublebond. A heteroalkenyl may optionally include more than one double bondand/or one or more triple bonds in additional to the one or more doublebonds. The term “heteroalkynyl,” by itself or in combination withanother term, means, unless otherwise stated, a heteroalkyl including atleast one triple bond. A heteroalkynyl may optionally include more thanone triple bond and/or one or more double bonds in additional to the oneor more triple bonds.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated,non-aromatic cyclic versions of “alkyl” and “heteroalkyl,” respectively,wherein the carbons making up the ring or rings do not necessarily needto be bonded to a hydrogen due to all carbon valencies participating inbonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,3-hydroxy-cyclobut-3-enyl-1,2, dione, 1H-1,2,4-triazolyl-5(4H)-one,4H-1,2,4-triazolyl, and the like. Examples of heterocycloalkyl include,but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A“cycloalkylene” and a “heterocycloalkylene,” alone or as part of anothersubstituent, means a divalent radical derived from a cycloalkyl andheterocycloalkyl, respectively. A heterocycloalkyl moiety may includeone ring heteroatom (e.g., O, N, S, Si, or P). A heterocycloalkyl moietymay include two optionally different ring heteroatoms (e.g., O, N, S,Si, or P). A heterocycloalkyl moiety may include three optionallydifferent ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkylmoiety may include four optionally different ring heteroatoms (e.g., O,N, S, Si, or P). A heterocycloalkyl moiety may include five optionallydifferent ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkylmoiety may include up to 8 optionally different ring heteroatoms (e.g.,O, N, S, Si, or P).

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed below. An “arylene” and a “heteroarylene,” alone or as part ofanother substituent, mean a divalent radical derived from an aryl andheteroaryl, respectively. Non-limiting examples of aryl and heteroarylgroups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl,indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl,pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl,quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl,benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl,pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl,furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl,benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl,diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl,pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl,or quinolyl. The examples above may be substituted or unsubstituted anddivalent radicals of each heteroaryl example above are non-limitingexamples of heteroarylene. A heteroaryl moiety may include one ringheteroatom (e.g., O, N, or S). A heteroaryl moiety may include twooptionally different ring heteroatoms (e.g., O, N, or S). A heteroarylmoiety may include three optionally different ring heteroatoms (e.g., O,N, or S). A heteroaryl moiety may include four optionally different ringheteroatoms (e.g., O, N, or S). A heteroaryl moiety may include fiveoptionally different ring heteroatoms (e.g., O, N, or S). An aryl moietymay have a single ring. An aryl moiety may have two optionally differentrings. An aryl moiety may have three optionally different rings. An arylmoiety may have four optionally different rings. A heteroaryl moiety mayhave one ring. A heteroaryl moiety may have two optionally differentrings. A heteroaryl moiety may have three optionally different rings. Aheteroaryl moiety may have four optionally different rings. A heteroarylmoiety may have five optionally different rings.

A fused ring heterocycloalkyl-aryl is an aryl fused to aheterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is aheteroaryl fused to a heterocycloalkyl. A fused ringheterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkylfused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl,fused ring heterocycloalkyl-heteroaryl, fused ringheterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substitutentsdescribed herein.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having theformula —S(O₂)—R′, where R′ is a substituted or unsubstituted alkylgroup as defined above. R′ may have a specified number of carbons (e.g.,“C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl”, “heteroalkyl”, “cycloalkyl”,“heterocycloalkyl”, “aryl”, and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C═(O)NR″NR′″R″″, —CN, —NO₂, in a number ranging from zeroto (2m′+1), where m′ is the total number of carbon atoms in suchradical. R, R′, R″, R′″, and R″″ each preferably independently refer tohydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted heteroaryl, substituted orunsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″, and R″″ group when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 4-, 5-, 6-, or 7-memberedring. For example, —NR′R″ includes, but is not limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C═(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system; and where R′, R″,R′″, and R″″ are preferably independently selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″, and R″″ groupswhen more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′— (C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,        —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,        —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃,        —OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl,        unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,        unsubstituted aryl, unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,            —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,            —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃,            —OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl,            unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,            unsubstituted aryl, unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,                —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,                —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H,                —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstituted alkyl,                unsubstituted heteroalkyl, unsubstituted cycloalkyl,                unsubstituted heterocycloalkyl, unsubstituted aryl,                unsubstituted heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, heteroaryl, substituted with at least one                substituent selected from: oxo, halogen, —CF₃, —CN, —OH,                —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,                —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H,                —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section, figures, or tables below.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19(1977)). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts. Otherpharmaceutically acceptable carriers known to those of skill in the artare suitable for the present invention. Salts tend to be more soluble inaqueous or other protonic solvents than are the corresponding free baseforms. In other cases, the preparation may be a lyophilized powder in 1mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Provided herein are agents (e.g. compounds, drugs, therapeutic agents)that may be in a prodrug form. Prodrugs of the compounds describedherein are those compounds that readily undergo chemical changes underselect physiological conditions to provide the final agents (e.g.compounds, drugs, therapeutic agents). Additionally, prodrugs can beconverted to agents (e.g. compounds, drugs, therapeutic agents) bychemical or biochemical methods in an ex vivo environment. Prodrugsdescribed herein include compounds that readily undergo chemical changesunder select physiological conditions to provide agents (e.g. compounds,drugs, therapeutic agents) to a biological system (e.g. in a subject).

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like)salts.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

In embodiments, a compound as described herein may include multipleinstances of R² and/or other variables. In such embodiments, eachvariable may optional be different and be appropriately labeled todistinguish each group for greater clarity. For example, where each R²is different, they may be referred to, for example, as R^(2.1), R^(2.2),R^(2.3), and/or R^(2.4) respectively, wherein the definition of R² isassumed by R^(2.1), R^(2.2), R^(2.3), and/or R^(2.4). The variables usedwithin a definition of R² and/or other variables that appear at multipleinstances and are different may similarly be appropriately labeled todistinguish each group for greater clarity. In some embodiments, thecompound is a compound described herein (e.g., in an aspect, embodiment,example, claim, table, scheme, drawing, or figure).

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls.

Where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different. For example, where a moiety hereinis R¹²-substituted or unsubstituted alkyl, a plurality of R¹²substituents may be attached to the alkyl moiety wherein each R¹²substituent is optionally different. Where an R-substituted moiety issubstituted with a plurality R substituents, each of the R-substituentsmay be differentiated herein using a prime symbol (′) such as R′, R″,etc. For example, where a moiety is R¹²-substituted or unsubstitutedalkyl, and the moiety is substituted with a plurality of R¹²substituents, the plurality of R¹² substituents may be differentiated asR¹²′, R¹²″, R¹²″′, etc. In embodiments, the plurality of R substituentsis 3. In embodiments, the plurality of R substituents is 2.

In embodiments, a compound as described herein may include multipleinstances of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴and/or other variables. In such embodiments, each variable may optionalbe different and be appropriately labeled to distinguish each group forgreater clarity. For example, where each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁹,R¹⁰, R¹¹, R¹², R¹³, and/or R¹⁴, is different, they may be referred to,for example, as R^(1.1), R^(1.2), R^(1.3), R^(1.4), R^(2.1), R^(2.2),R^(2.3), R^(2.4), R^(3.1), R^(3.2), R^(3.3), R^(3.4), R^(4.1), R^(4.2),R^(4.3), R^(4.4), R^(5.1), R^(5.2), R^(5.3), R^(5.4), R^(6.1), R^(6.2),R^(6.3), R^(6.4), R^(7.1), R^(7.2), R^(7.3), R^(7.4), R^(9.1), R^(9.2),R^(9.3), R^(9.4), R^(10.1), R^(10.2), R^(10.3), R^(10.4), R^(11.1),R^(11.2), R^(11.3), R^(11.4), R^(12.1), R^(12.2), R^(12.3), R^(12.4),R^(13.1), R^(13.2), R^(13.3), R^(13.4), R^(14.1), R^(14.2), R^(14.3),and/or R^(14.4), respectively, wherein the definition of R¹ is assumedby R^(1.1), R^(1.2), R^(1.3), and/or R^(1.4), the definition of R² isassumed by R^(2.1), R^(2.2), R^(2.3), R^(2.4), the definition of R³ isassumed by R^(3.1), R^(3.2), R^(3.3), and/or R^(3.4), the definition ofR⁴ is assumed by R^(4.1), R^(4.2), R^(4.3), and/or R^(4.4), thedefinition of R⁵ is assumed by R^(5.1), R^(5.2), R^(5.3), and/orR^(5.4), the definition of R⁶ is assumed by R^(6.1), R^(6.2), R^(6.3),and/or R^(6.4), the definition of R⁷ is assumed by R^(7.1), R^(7.2),R^(7.3), and/or R^(7.4), the definition of R⁹ is assumed by R^(9.1),R^(9.2), R^(9.3), and/or R^(9.4), the definition of R¹⁰ is assumed byR^(10.1), R^(10.2), R^(10.3), and/or R^(10.4), the definition of R¹¹ isassumed by R^(11.1), R^(11.2), R^(11.3), and/or R^(11.4), the definitionof R¹² is assumed by R^(12.1), R^(12.2), R^(12.3), and/or R^(12.4), thedefinition of R¹³ is assumed by R^(13.1), R^(13.2), R^(13.3), and/orR^(13.4), the definition of R¹⁴ is assumed by R^(14.1), R^(14.2),R^(14.3), and/or R^(14.4). The variables used within a definition of R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰, R¹¹, R¹², R¹³, and/or R¹⁴, and/or othervariables that appear at multiple instances and are different maysimilarly be appropriately labeled to distinguish each group for greaterclarity.

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Antibodies are large, complex molecules (molecular weight of 150,000 orabout 1320 amino acids) with intricate internal structure. A naturalantibody molecule contains two identical pairs of polypeptide chains,each pair having one light chain and one heavy chain. Each light chainand heavy chain in turn consists of two regions: a variable (“V”) regioninvolved in binding the target antigen, and a constant (“C”) region thatinteracts with other components of the immune system. The light andheavy chain variable regions come together in 3-dimensional space toform a variable region that binds the antigen (for example, a receptoron the surface of a cell). Within each light or heavy chain variableregion, there are three short segments (averaging 10 amino acids inlength) called the complementarity determining regions (“CDRs”). The sixCDRs in an antibody variable domain (three from the light chain andthree from the heavy chain) fold up together in 3-dimensional space toform the actual antibody binding site which docks onto the targetantigen. The position and length of the CDRs have been precisely definedby Kabat, E. et al., Sequences of Proteins of Immunological Interest,U.S. Department of Health and Human Services, 1983, 1987. The part of avariable region not contained in the CDRs is called the framework(“FR”), which forms the environment for the CDRs.

The term “antibody” is used according to its commonly known meaning inthe art. As used herein, “antibody” may also refer to the antigenbinding fragment thereof. Antibodies exist, e.g., as intactimmunoglobulins or as a number of well-characterized fragments producedby digestion with various peptidases. Thus, for example, pepsin digestsan antibody below the disulfide linkages in the hinge region to produceF(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H1) by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region, therebyconverting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially Fab with part of the hinge region (see FundamentalImmunology (Paul ed., 3d ed. 1993). While various antibody fragments aredefined in terms of the digestion of an intact antibody, one of skillwill appreciate that such fragments may be synthesized de novo eitherchemically or by using recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies, or those synthesizedde novo using recombinant DNA methodologies (e.g., single chain Fv) orthose identified using phage display libraries (see, e.g., McCafferty etal., Nature 348:552-554 (1990)).

A single-chain variable fragment (scFv) is typically a fusion protein ofthe variable regions of the heavy (VH) and light chains (VL) ofimmunoglobulins, connected with a short linker peptide of 10 to about 25amino acids. The linker may usually be rich in glycine for flexibility,as well as serine or threonine for solubility. The linker can eitherconnect the N-terminus of the VH with the C-terminus of the VL, or viceversa.

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole etal., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).“Monoclonal” antibodies (mAb) refer to antibodies derived from a singleclone. Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptidesof this invention. Also, transgenic mice, or other organisms such asother mammals, may be used to express humanized antibodies.Alternatively, phage display technology can be used to identifyantibodies and heteromeric Fab fragments that specifically bind toselected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

The epitope of a mAb is the region of its antigen to which the mAbbinds. Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1×, 5×, 10×, 20× or 100× excess of one antibody inhibitsbinding of the other by at least 30% but preferably 50%, 75%, 90% oreven 99% as measured in a competitive binding assay (see, e.g., Junghanset al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies havethe same epitope if essentially all amino acid mutations in the antigenthat reduce or eliminate binding of one antibody reduce or eliminatebinding of the other. Two antibodies have overlapping epitopes if someamino acid mutations that reduce or eliminate binding of one antibodyreduce or eliminate binding of the other.

For preparation of suitable antibodies of the invention and for useaccording to the invention, e.g., recombinant, monoclonal, or polyclonalantibodies, many techniques known in the art can be used (see, e.g.,Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., ImmunologyToday 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual(1988); and Goding, Monoclonal Antibodies: Principles and Practice (2ded. 1986)). The genes encoding the heavy and light chains of an antibodyof interest can be cloned from a cell, e.g., the genes encoding amonoclonal antibody can be cloned from a hybridoma and used to produce arecombinant monoclonal antibody. Gene libraries encoding heavy and lightchains of monoclonal antibodies can also be made from hybridoma orplasma cells. Random combinations of the heavy and light chain geneproducts generate a large pool of antibodies with different antigenicspecificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques forthe production of single chain antibodies or recombinant antibodies(U.S. Pat. Nos. 4,946,778, 4,816,567) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedor human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); andLonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art (e.g., U.S. Pat. Nos. 4,816,567; 5,530,101; 5,859,205;5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and6,329,511; WO 87/02671; EP Patent Application 0173494; Jones et al.(1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534).Humanized antibodies are further described in, e.g., Winter and Milstein(1991) Nature 349:293. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.These non-human amino acid residues are often referred to as importresidues, which are typically taken from an import variable domain.Humanization can be essentially performed following the method of Winterand co-workers (see, e.g., Morrison et al., PNAS USA, 81:6851-6855(1984), Jones et al., Nature 321:522-525 (1986); Riechmann et al.,Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92(1988), Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489-498(1991); Padlan, Molec. Immun., 31(3):169-217 (1994)), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such humanized antibodies are chimeric antibodies(U.S. Pat. No. 4,816,567), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies. For example, polynucleotides comprising a first sequencecoding for humanized immunoglobulin framework regions and a secondsequence set coding for the desired immunoglobulin complementaritydetermining regions can be produced synthetically or by combiningappropriate cDNA and genomic DNA segments. Human constant region DNAsequences can be isolated in accordance with well known procedures froma variety of human cells.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity. The preferred antibodies of, and for useaccording to the invention include humanized and/or chimeric monoclonalantibodies.

Techniques for conjugating therapeutic agents to antibodies are wellknown (see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery” inControlled Drug Delivery (2^(nd) Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)). As used herein, the term “antibody-drug conjugate” or “ADC”refers to a therapeutic agent conjugated or otherwise covalently boundto an antibody. A “therapeutic agent” as referred to herein, is acomposition useful in treating or preventing a disease such as cancer.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies can be selectedto obtain only a subset of antibodies that are specificallyimmunoreactive with the selected antigen and not with other proteins.This selection may be achieved by subtracting out antibodies thatcross-react with other molecules. A variety of immunoassay formats maybe used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual(1998) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

A “ligand” refers to an agent, e.g., a polypeptide or other molecule,capable of binding to a receptor.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be, forexample, a pharmaceutical composition as provided herein and a cell or apCREB detection agent as described herein and a pCREB antigen. Inembodiments contacting includes, for example, allowing a pharmaceuticalcomposition as described herein to interact with a cell or a patient. Infurther embodiments, contacting includes, for example, allowing a pCREBdetection agent as described herein to interact with a pCREB antigen.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may In embodiments be conjugated to a moiety thatdoes not consist of amino acids. The terms apply to amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers. A “fusion protein” refers to a chimeric proteinencoding two or more separate protein sequences that are recombinantlyexpressed as a single moiety.

The term “peptidyl” and “peptidyl moiety” means a monovalent peptide.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% identity over a specified region, e.g., of the entire polypeptidesequences of the invention or individual domains of the polypeptides ofthe invention), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Optionally, the identity exists over a region that is at leastabout 50 nucleotides in length, or more preferably over a region that is100 to 500 or 1000 or more nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of, e.g., a full length sequence or from 20 to 600, about 50to about 200, or about 100 to about 150 amino acids or nucleotides inwhich a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well-knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “isolated,” when applied to a protein, denotes that the proteinis essentially free of other cellular components with which it isassociated in the natural state. It is preferably in a homogeneous statealthough it can be in either a dry or aqueous solution. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. The term“purified” denotes that a protein gives rise to essentially one band inan electrophoretic gel. Particularly, it means that the protein is atleast 85% pure, more preferably at least 95% pure, and most preferablyat least 99% pure.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor (e.g., an A2A receptorantagonist or a PD-1 signaling pathway inhibitor) interaction meansnegatively affecting (e.g., decreasing) the activity or function of theprotein (e.g., decreasing the activity of an A2A receptor or a PD-1protein or PD-L1 protein) relative to the activity or function of theprotein in the absence of the inhibitor (e.g., an A2A receptorantagonist or a PD-1 signaling pathway inhibitor). In some embodiments,inhibition refers to reduction of a disease or symptoms of disease(e.g., cancer). Thus, inhibition includes, at least in part, partiallyor totally blocking stimulation, decreasing, preventing, or delayingactivation, or inactivating, desensitizing, or down-regulating signaltransduction or enzymatic activity or the amount of a protein (e.g., anA2A receptor or a PD-1 protein or PD-L1 protein). Similarly an“inhibitor” is a compound or protein that inhibits an A2A receptor or aPD-1 protein or PD-L1 protein, e.g., by binding, partially or totallyblocking, decreasing, preventing, delaying, inactivating, desensitizing,or down-regulating activity (e.g., an A2A receptor activity or a PD-1protein activity or PD-L1 protein activity).

An “anti-cancer agent” is a therapeutic used in the treatment orprevention of cancer. An anti-cancer agent can be a large or smallmolecule. Example anti-cancer agents include antibodies, smallmolecules, and large molecules or combinations thereof.

“Anti-cancer agent” is used in accordance with its plain ordinarymeaning and refers to a composition (e.g. compound, drug, antagonist,inhibitor, modulator) having antineoplastic properties or the ability toinhibit the growth or proliferation of cells. In some embodiments, ananti-cancer agent is a chemotherapeutic. In some embodiments, ananti-cancer agent is an agent identified herein having utility inmethods of treating cancer. In some embodiments, an anti-cancer agent isan agent approved by the FDA or similar regulatory agency of a countryother than the USA, for treating cancer. Examples of anti-cancer agentsinclude, but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2)inhibitors (e.g. XL518, CI-1040, PD035901, selumetinib/AZD6244,GSK1120212/trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901,U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylatingagents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan,melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogenmustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil,meiphalan), ethylenimine and methylmelamines (e.g., hexamethlymelamine,thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine, lomusitne, semustine, streptozocin), triazenes(decarbazine)), anti-metabolites (e.g., 5-azathioprine, leucovorin,capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folicacid analog (e.g., methotrexate), or pyrimidine analogs (e.g.,fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g.,vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin,paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g.,irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate,teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin,daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), platinum-based compounds or platinumcontaining agents (e.g. cisplatin, oxaloplatin, carboplatin),anthracenedione (e.g., mitoxantrone), substituted urea (e.g.,hydroxyurea), methyl hydrazine derivative (e.g., procarbazine),adrenocortical suppressant (e.g., mitotane, aminoglutethimide),epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin,doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors ofmitogen-activated protein kinase signaling (e.g. U0126, PD98059,PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006,wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies(e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, alltrans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL), 5-aza-2′-deoxycytidine, all transretinoic acid, doxorubicin, vincristine, etoposide, gemcitabine,imatinib (Gleevec®), geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352,20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-b enzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenyl acetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatinstimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; iimofosine; interleukin I1 (includingrecombinant interleukin II, or r1L.sub.2), interferon alfa-2a;interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferonbeta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride;lanreotide acetate; letrozole; leuprolide acetate; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie;nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride, agents that arrest cells in the G2-M phases and/ormodulate the formation or stability of microtubules, (e.g. Taxol™ (i.e.paclitaxel), Taxotere™, compounds comprising the taxane skeleton,Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128),Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829,Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010),Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g.Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4,Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, andSpongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 andNSC-D-669356), Epothilones (e.g. Epothilone A, Epothilone B, EpothiloneC (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB,and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone BN-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B(i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F anddEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin(i.e. TZT-1027), Vincristine sulfate, Cryptophycin 52 (i.e. LY-355703),Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e. NSC-106969),Oncocidin A1 (i.e. BTO-956 and DIME), Fijianolide B, Laulimalide,Narcosine (also known as NSC-5366), Nascapine, Hemiasterlin, Vanadoceneacetylacetonate, Monsatrol, lnanocine (i.e. NSC-698666), Eleutherobins(such as Desmethyleleutherobin, Desaetyleleutherobin, lsoeleutherobin A,and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B,Diazonamide A, Taccalonolide A, Diozostatin, (−)-Phenylahistin (i.e.NSCL-96F037), Myoseverin B, Resverastatin phosphate sodium, steroids(e.g., dexamethasone), finasteride, aromatase inhibitors,gonadotropin-releasing hormone agonists (GnRH) such as goserelin orleuprolide, adrenocorticosteroids (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate, megestrol acetate, medroxyprogesteroneacetate), estrogens (e.g., di ethlystilbestrol, ethinyl estradiol),antiestrogen (e.g., tamoxifen), androgens (e.g., testosteronepropionate, fluoxymesterone), antiandrogen (e.g., flutamide),immunostimulants (e.g., Bacillus Calmette-Guérin (BCG), levamisole,interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g.,anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonalantibodies), immunotoxins (e.g., anti-CD33 monoclonalantibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y or ¹³¹I,etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,epirubicin, topotecan, itraconazole, vindesine, cerivastatin,vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib,gefitinib, EGFR inhibitors, epidermal growth factor receptor(EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa™),erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™),panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992,CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306,ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethylerlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002,WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib,sunitinib, dasatinib, hormonal therapies, or the like.

“Analog” and “analogue” are used interchangeably and are used inaccordance with their plain ordinary meaning within Chemistry andBiology and refers to a chemical compound that is structurally similarto another compound (i.e., a so-called “reference” compound) but differsin composition, e.g., in the replacement of one atom by an atom of adifferent element, or in the presence of a particular functional group,or the replacement of one functional group by another functional group,or the absolute stereochemistry of one or more chiral centers of thereference compound, including isomers thereof. Accordingly, an analog isa compound that is similar or comparable in function and appearance butnot in structure or origin to a reference compound. In embodiments, ananalog is an adenosine analog.

An example adenosine analog is 5′-N-ethylcarboxamido-adenosine (NECA),having the structure shown below:

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments,about means within a standard deviation using measurements generallyacceptable in the art. In embodiments, about means a range extending to+/−10% of the specified value. In embodiments, about means the specifiedvalue.

An “A2A receptor” or “adenosine A2A receptor” as referred to hereinincludes any of the recombinant or naturally-occurring forms of theadenosine A2A receptor also known as ADORA2A or variants or homologsthereof that maintain adenosine A2A receptor activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto adenosine A2A receptor). In some aspects, the variants or homologshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring adenosine A2A receptor. In embodiments, theadenosine A2A receptor is substantially identical to the proteinidentified by the UniProt reference number P29274 or a variant orhomolog having substantial identity thereto. In embodiments, theadenosine A2A receptor is substantially identical to the proteinidentified by the UniProt reference number Q60613 or a variant orhomolog having substantial identity thereto.

“A2B receptor” or “A2BR” or “Adenosine A_(2B) receptor” are usedinterchangeably. A2B receptors are expressed in some mast cells, such asthe BR line of canine mastocytoma cells, which appear to be responsiblefor triggering acute Ca²⁺ mobilization and degranulation. (SeeAuchampach et al., Mol. Pharmacol. 1997, 52, 846-S60 and Forsyth et al.,Inflamm. Res. 1999, 48, 301-307.) Adenosine A_(2B) receptors alsotrigger Ca²⁺ mobilization, and participate in a delayed IL8 release fromhuman HMC-1 mast cells. Other functions associated with the A_(2B) ARare the control of cell growth and gene expression, (See Neary et al.,Trends Neurosci. 1996, 19, 13-18.) endothelial-dependent vasodilation(See Martin et al., J Pharmacol. Exp. Ther. 1993, 265, 248-2, 53.), andfluid secretion from intestinal epithelia. (See Strohmeier, et al., JBiol. Chem. 1995, 270, 2387-2394.) Adenosine acting through A_(2B)receptor subtype has also been reported to stimulate chloridepermeability in cells expressing the cystic fibrosis transportregulator. (See Clancy et al., Am. J Physiol. 1999, 276, C361-C369.)Example A2b receptor antagonists are described in WO 2008002902,included herein by reference in its entirety. In embodiments, theadenosine A2b receptor is substantially identical to the proteinidentified by the UniProt reference number P29275 or a variant orhomolog having substantial identity thereto.

“Adenosine receptor agonist” refers to a molecule that activatesadenosine receptors (e.g. A2a or A2b receptors). Adenosine receptorsagonists can be small or large molecule agonists. Example adenosinereceptors agonists include adenosine, NECA, or analogs thereof.

“Adenosine receptor antagonist” references to a molecule that inhibitsactivity of adenosine receptors (e.g. A2a or A2b receptors). Adenosinereceptors antagonists can be small or large molecule antagonists. Inembodiments, CPI-444 is an example A2A receptor antagonist.

A “PD-1 protein” or “PD-1” as referred to herein includes any of therecombinant or naturally-occurring forms of the Programmed cell deathprotein 1 (PD-1) also known as cluster of differentiation 279 (CD 279)or variants or homologs thereof that maintain PD-1 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to PD-1 protein). In some aspects, the variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring PD-1 protein. In embodiments, the PD-1 protein issubstantially identical to the protein identified by the UniProtreference number Q15116 or a variant or homolog having substantialidentity thereto. In embodiments, the PD-1 protein is substantiallyidentical to the protein identified by the UniProt reference numberQ02242 or a variant or homolog having substantial identity thereto.

A “pCREB protein” or “pCREB” as referred to herein includes any of therecombinant or naturally-occurring forms of the cAMP responseelement-binding protein (pCREB) or variants or homologs thereof thatmaintain pCREB activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to pCREB protein). In someaspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to a naturally occurring pCREB protein. Inembodiments, the pCREB protein is substantially identical to the proteinidentified by the UniProt reference number P16220 or a variant orhomolog having substantial identity thereto.

A “PD-1 protein” or “PD-1” as referred to herein includes any of therecombinant or naturally-occurring forms of the Programmed cell deathprotein 1 (PD-1) also known as cluster of differentiation 279 (CD 279)or variants or homologs thereof that maintain PD-1 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to PD-1 protein). In some aspects, the variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring PD-1 protein. In embodiments, the PD-1 protein issubstantially identical to the protein identified by the UniProtreference number Q15116 or a variant or homolog having substantialidentity thereto. In embodiments, the PD-1 protein is substantiallyidentical to the protein identified by the UniProt reference numberQ02242 or a variant or homolog having substantial identity thereto.

The term “atezolizumab” or “MPDL3280A” refers to a fully humanized,engineered monoclonal antibody of IgG1 isotype against the proteinprogrammed cell death ligand 1 (PD-L1). In the customary sense,atezolizumab refers to CAS Registry number 1380723-44-3. Atezolizumabmay be referred to as an anti-cancer agent. In embodiments, atezolizumabis referred to as with the tradename Tecentriq®.

Cyclic AMP (cAMP) response element binding protein (CREB) is a cellulartranscription factor. CREB is activated by signaling cascades resultantfrom an array of extracellular signals. One such activating signalcascade is triggered by agonist binding to adenosine receptor (e.g. A2Aand A2B receptors). Agonist activation of adenosine receptor results inactivation of CREB by phosphorylation. Agonist activation of adenosinereceptor also results in activation of protein kinase A (PKA) upstreamof CREB. In embodiments, CREB is substantially identical to the proteinidentified by the UniProt reference number P16220 or a variant orhomolog having substantial identity thereto. In embodiments, CREB isphosphorylated at Serine 133.

A “pCREB detection agent” refers to a chemical or molecular moietycapable of identifying phosphorylated CREB. A pCREB detection agent maycomprise an antibody or other specific for the phosphorylated CREB.Antibodies specific to pCREB are commercially available, for examplep-CREB Antibody (Ser133) (Cell Signaling Technology Cat. No.: 14001 orSanta Cruz Biotechnology Cat. No.: sc-7978).

The term “blood cell detection agent” refers to a chemical or molecularmoiety capable of identifying blood cells. A blood cell detection agentcan refer to, for example, a chemical stain or an antibody against cellsurface markers. Example blood cell detection agents include B celldetection agents and T cell detection agents.

The term “B cell detection agent” refers to a chemical or molecularmoiety capable of identifying B cells. In examples, a B cell detectionagent can be an antibody to a B cell specific surface maker (e.g. anantibody against CD19, or an antibody against CD20). B cell detectionagents can be used alone or in combination. B cell detection agents canfurther be detected by fluorescence activated cell sorting (FACS).

“B Cells” or “B lymphocytes” refer to their standard use in the art. Bcells are lymphocytes, a type of white blood cell (leukocyte), thatdevelops into a plasma cell (a “mature B cell”), which producesantibodies. An “immature B cell” is a cell that can develop into amature B cell. Generally, pro-B cells undergo immunoglobulin heavy chainrearrangement to become pro B pre B cells, and further undergoimmunoglobulin light chain rearrangement to become an immature B cells.Immature B cells include T1 and T2 B cells.

“T cell detection agents” refers to a chemical or molecular moietycapable of identifying T cells. In examples, a T cell detection agentcan be an antibody to a T cell specific surface maker (e.g. an antibodyagainst CD3, and antibody against C4, or an antibody against CD8). Tcell detection agents can be used alone or in combination. T celldetection agents can further be detected by fluorescence activated cellsorting (FACS).

“T cells” or “T lymphocytes” as used herein are a type of lymphocyte (asubtype of white blood cell) that plays a central role in cell-mediatedimmunity. They can be distinguished from other lymphocytes, such as Bcells and natural killer cells, by the presence of a T-cell receptor onthe cell surface. T cells include, for example, natural killer T (NKT)cells, cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, and Thelper cells. Different types of T cells can be distinguished by use ofT cell detection agents.

The term “cell subset detection agent” refers to a chemical or moleculedetection agent that can be used to identify and distinguish a specificsubset of cells (e.g. senescent cells, naïve cells, effector cells,memory cells etc). Example cell subset detection agents include “naïvecell detection agents”, “memory cell detection agents”, and “effectorcell detection agent.” Cell subset detection agents can includeantibodies against distinguishing cell surface markers. In embodiments,cell subset detection agents include antibodies against CD27 orantibodies against CD45RA.

The term “apoptotic cells detection agent” refers to a chemical ormolecule detection agent that can be used to identify and distinguishapoptotic cells. Apoptotic cell detection agents can include antibodiesagainst distinguishing cell surface markers. An example apoptosisdetection agent includes an antibody against cPARP. PARP is inactivatedby caspase cleavage. Cleaved poly-ADP-ribose polymerase (PARP) (cPARP)is the cleavage product of PARP. cPARP can be used as a marker forapoptosis.

The term “CD” or “Cluster of Differentiation” refers to a nomenclaturesystem for antigens found on lymphocytes, although CD antigens can befound on cells other than lymphocytes. This nomenclature is used to nameantigens recognized by monoclonal antibodies that specifically bind anantigen on B cells, T cells or antigen presenting cells. Each numericantigen is a specific protein that is recognized in the art by its CDdesignation.

The term “CD3” as referred to herein is a protein complex comprisingfour chains including a CD3γ chain, a CD3δ chain, and two CD3ε chains.An example sequences of CD3 complex chains include: Epsilon chainprecursor (GENBANK® Accession No. NP_000724.1); Gamma chain precursor(GENBANK® Accession No. NP_000064.1); Delta chain precursor (GENBANK®Accession No. NP_000723.1) which are incorporated herein by reference.Multiple isoforms are possible for each of the chains of CD3.

The term “CD4” as referred to herein is a glycoprotein expressed on thesurface of T helper cells, regulatory T cells, monocytes, macrophages,and dendritic cells. CD4 was originally known as leu-3 and T4 (after theOKT4 monoclonal antibody). CD4 as referred to herein has fourimmunoglobulin domains (D₁ to D₄) that are exposed on the extracellularsurface of the cell, see ENTREZ No. 920, UNIPROT No. P01730, andGENBANK® Accession No. NP_000607, which are incorporated by reference.

A “CD4⁺ T lymphocyte” or “CD4 T cell” as referred to herein islymphocyte that expresses the CD4 glycoprotein on its surface. CD4 Tcells include helper T cells, which are T cells that help orchestratethe immune response, including antibody responses and killer T cellresponses. CD4 T cell precursors differentiate into one of severalsubtypes, including TH1 (type 1 helper T cell), TH2 (type 2 helper Tcell), TH3 (T helper 3 cells), TH17 (T helper 17 cells) or TFH(Follicular B helper T cells). These subtypes of helper T cells arecharacterized by their secretion of different cytokines to facilitatedifferent types of immune responses. In embodiments, a CD4 T cell is aneffector T cell. An “effector T cell” as referred to herein is a T cellthat has been activated by its cognate antigen, and is actively involvedin eliminating a pathogen. Thus, an effector T cell actively responds toa stimulus (a pathogen or a co-stimulation) and carries out acell-mediated immune response. Non-limiting examples of effector T cellsas referred to herein include helper T cells, killer T cells (cytotoxicT cells) and regulatory T cells.

The term “CD8” as referred to herein is a transmembrane glycoproteinthat serves as a co-receptor for the T cell receptor (TCR). Like theTCR, CD8 binds to a major histocompatibility complex (MHC) molecule, butis specific for the class I MHC protein, see ENTREZ No. 925 and UNIPROTNo. P01732, which are incorporated by reference herein.

A “CD8⁺ T lymphocyte” or “CD8 T cell” as referred to herein is alymphocyte that expresses the CD8 glycoprotein on its surface. Examplesof CD8 T cells include cytotoxic T cells and natural killer cells. Inone embodiment, a CD8 T cell is a cytotoxic T cell. In embodiments, aCD8 T cell is a suppressor T cell.

CD20 is involved in regulating early steps in the activation anddifferentiation process of B cells (Tedder et al., Eur. J. Immunol.16:881-887, 1986) and can function as a calcium ion channel (Tedder etal., J. Cell. Biochem. 14D:195, 1990). Exemplary amino acid sequencesfor CD19 are provided in GENBANK® Accession Nos. NP_068769.2 (human),NP_690605.1 (human), and NP_031667.1 (mouse), which are incorporated byreference herein.

CD27: A costimulatory immune checkpoint molecule. CD27 precursor(human)(GENBANK® Accession No. NP_001233.1). Multiple isoforms exist.

The term “CD45RA” as provided herein refers to the CD45 Receptor antigenalso known as Protein tyrosine phosphatase, receptor type, C (PTPRC).Exemplary amino acid sequences for CD45RA include GENBANK® AccessionNos. NP_002829.3, NP_563578.2, NP_563578.2, and NP_002829.3, which areall incorporated herein by reference. CD45RA is expressed on naïve Tcells, as well as on CD8- and CD4-expressing effector cells. Afterantigen interaction, T cells gain expression of CD45RO and loseexpression of CD45RA. Thus, either CD45RA or CD45RO is used to generallydifferentiate the naïve from memory T cell populations. Thus, a“CD45RA-negative CD8 T cell” as provided herein is a CD8 T cell whichlacks expression of detectable amounts of CD45RA. In embodiments, theCD45RA-negative CD8 T cell is a memory T cell. A “CD45RA-negative CD4 Tcell” as provided herein is a CD4 T cell which lacks expression ofdetectable amounts of CD45RA. In embodiments, the CD45RA-negative CD4 Tcell is a memory T cell. In embodiments, the CD45RA-negative CD8 T cellis a memory T cell.

A “memory T cell” is a T cell that has previously encountered andresponded to its cognate antigen during prior infection, encounter withcancer or previous vaccination. At a second encounter with its cognateantigen memory T cells can reproduce (divide) to mount a faster andstronger immune response than the first time the immune system respondedto the pathogen. In embodiments, the memory T cell is a CD45RA-negativeCD4 T cell. In embodiments, the memory T cell is a CD45RA-negative CD8 Tcell.

A “regulatory T cell” or “suppressor T cell” is a lymphocyte whichmodulates the immune system, maintains tolerance to self-antigens, andprevents autoimmune disease. Regulatory T cells express the CD4, FOXP3,and CD25 and are thought to be derived from the same lineage as naïveCD4 cells.

A “fixation agent” is a chemical or molecular agent capable of fixing acell (e.g. of preserving a cell). A fixation agent can be used toprevent further biological process in preparation for cell staining,imaging or sorting. Fixation agents can be used alone or in combination.Non-limiting examples of fixation agents include formaldehyde,glutaraldehyde, ethanol, methanol, Potassium dichromate, chromic acid,and potassium permanganate, B-5, Zenker's fixative, picrates, and HOPE.

A “cell permeabilizing agent” can include chemical or molecular agent,or a mechanical means of permeabilizing a cell. Non-limiting examples ofpermeabilization agents include organic solvents, such as methanol andacetone, and detergents such as saponin, Triton X-100 and Tween-20. Theorganic solvents dissolve lipids from cell membranes making thempermeable to antibodies.

A “refractory subject” as provided herein is a subject that has been oris being treated for a disease or condition and does not respond toattempted forms of treatment for said disease or condition. For example,a cancer is said to be refractory when it does not respond to (or isresistant to) cancer treatment. A refractory cancer is also known asresistant cancer. Thus, a refractory subject is a subject that does notrespond or is resistant to treatment of a disease or condition thesubject is suffering from. In embodiments, a refractory subject is acancer patient unresponsive to anti-PD-1 therapy. Where the cancerpatient is unresponsive to anti-PD-1 therapy the patient shows less than20% reduction in tumor size or volume after administration of anti-PD-1relative to a control. Thus, in embodiments, a refractory subject showsless than 20% reduction in tumor size or volume after administration ofanti-PD-1 relative to a control. In embodiments, a refractory subjectshows less than 10% reduction in tumor size or volume afteradministration of anti-PD-1 relative to a control. In embodiments, arefractory subject shows less than 5% reduction in tumor size or volumeafter administration of anti-PD-1 relative to a control. In embodiments,a refractory subject shows less than 1% reduction in tumor size orvolume after administration of anti-PD-1 relative to a control. Inembodiments, a refractory subject shows less than 0.5% reduction intumor size or volume after administration of anti-PD-1 relative to acontrol. In embodiments, a refractory subject shows less than 0.1%reduction in tumor size or volume after administration of anti-PD-1relative to a control.

The term “anti-tumor immune memory” as provided herein refers to theability of the immune system of a subject to recognize (memorize)previously encountered tumor antigen. Once the tumor antigen has beenrecognized, the immune system reproduces (e.g., through T cellactivation and proliferation) and can mount a faster and stronger immuneresponse than the first time it responded to the same tumor antigen.

The term “global immune activation” as provided herein refers to theactivation of immune cells of the adaptive immune system in a subject.Examples of immune cells activated during global immune activation arewithout limitation, antigen presenting cells (macrophages, dendriticcells), B cells and T cells. The activation may occur throughrecognition of a previously encountered antigen (tumor antigen) or itmay occur through encounter of a novel (not previously encountered)antigen (tumor antigen).

The terms “disease” or “condition” refer to a state of being or healthstatus of a patient or subject capable of being treated with a compound,pharmaceutical composition, or method provided herein. In embodiments,the disease is cancer (e.g. lung cancer, ovarian cancer, osteosarcoma,bladder cancer, cervical cancer, liver cancer, kidney cancer, skincancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia,lymphoma, head and neck cancer, colorectal cancer, prostate cancer,pancreatic cancer, melanoma, breast cancer, neuroblastoma). The diseasemay be an autoimmune, inflammatory, cancer, infectious, metabolic,developmental, cardiovascular, liver, intestinal, endocrine,neurological, or other disease.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a patient suspected ofhaving a given disease (cancer) and compared to samples from a knowncancer patient, or a known normal (non-disease) individual. A controlcan also represent an average value gathered from a population ofsimilar individuals, e.g., cancer patients or healthy individuals with asimilar medical background, same age, weight, etc. A control value canalso be obtained from the same individual, e.g., from anearlier-obtained sample, prior to disease, or prior to treatment. One ofskill will recognize that controls can be designed for assessment of anynumber of parameters.

One of skill in the art will understand which controls are valuable in agiven situation and be able to analyze data based on comparisons tocontrol values. Controls are also valuable for determining thesignificance of data. For example, if values for a given parameter arewidely variant in controls, variation in test samples will not beconsidered as significant.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals, including leukemias,lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas.Exemplary cancers that may be treated with a compound, pharmaceuticalcomposition, or method provided herein include lymphoma, sarcoma,bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer,esophageal cancer, gastric cancer, head and neck cancer, kidney cancer,myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g.triple negative, ER positive, ER negative, chemotherapy resistant,herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifenresistant, ductal carcinoma, lobular carcinoma, primary, metastatic),ovarian cancer, pancreatic cancer, liver cancer (e.g. hepatocellularcarcinoma), lung cancer (e.g. non-small cell lung carcinoma, squamouscell lung carcinoma, adenocarcinoma, large cell lung carcinoma, smallcell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme,glioma, melanoma, prostate cancer, castration-resistant prostate cancer,breast cancer, triple negative breast cancer, glioblastoma, ovariancancer, lung cancer, squamous cell carcinoma (e.g., head, neck, oresophagus), colorectal cancer, leukemia, acute myeloid leukemia,lymphoma, B cell lymphoma, or multiple myeloma. Additional examplesinclude, cancer of the thyroid, endocrine system, brain, breast, cervix,colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung,melanoma, mesothelioma, ovary, sarcoma, stomach, uterus orMedulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiplemyeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,primary brain tumors, cancer, malignant pancreatic insulanoma, malignantcarcinoid, urinary bladder cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, endometrial cancer,adrenal cortical cancer, neoplasms of the endocrine or exocrinepancreas, medullary thyroid cancer, medullary thyroid carcinoma,melanoma, colorectal cancer, papillary thyroid cancer, hepatocellularcarcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, LobularCarcinoma, Ductal Carcinoma, cancer of the pancreatic stellate cells,cancer of the hepatic stellate cells, or prostate cancer.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). Exemplary leukemias that may be treated with a compound,pharmaceutical composition, or method provided herein include, forexample, acute nonlymphocytic leukemia, chronic lymphocytic leukemia,acute granulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,aleukocythemic leukemia, basophylic leukemia, blast cell leukemia,bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, or undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a compound, pharmaceuticalcomposition, or method provided herein include a chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcomaof B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, or telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas that may betreated with a compound, pharmaceutical composition, or method providedherein include, for example, acral-lentiginous melanoma, amelanoticmelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, nodular melanoma, subungal melanoma, or superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acompound, pharmaceutical composition, or method provided herein include,for example, medullary thyroid carcinoma, familial medullary thyroidcarcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma,adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenalcortex, alveolar carcinoma, alveolar cell carcinoma, basal cellcarcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamouscell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,bronchogenic carcinoma, cerebriform carcinoma, cholangiocellularcarcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma,corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinomacutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductcarcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lobularcarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinomavillosum.

As used herein, the terms “metastasis,” “metastatic,” and “metastaticcancer” can be used interchangeably and refer to the spread of aproliferative disease or disorder, e.g., cancer, from one organ oranother non-adjacent organ or body part. Cancer occurs at an originatingsite, e.g., breast, which site is referred to as a primary tumor, e.g.,primary breast cancer. Some cancer cells in the primary tumor ororiginating site acquire the ability to penetrate and infiltratesurrounding normal tissue in the local area and/or the ability topenetrate the walls of the lymphatic system or vascular systemcirculating through the system to other sites and tissues in the body. Asecond clinically detectable tumor formed from cancer cells of a primarytumor is referred to as a metastatic or secondary tumor. When cancercells metastasize, the metastatic tumor and its cells are presumed to besimilar to those of the original tumor. Thus, if lung cancermetastasizes to the breast, the secondary tumor at the site of thebreast consists of abnormal lung cells and not abnormal breast cells.The secondary tumor in the breast is referred to a metastatic lungcancer. Thus, the phrase metastatic cancer refers to a disease in whicha subject has or had a primary tumor and has one or more secondarytumors. The phrases non-metastatic cancer or subjects with cancer thatis not metastatic refers to diseases in which subjects have a primarytumor but not one or more secondary tumors. For example, metastatic lungcancer refers to a disease in a subject with or with a history of aprimary lung tumor and with one or more secondary tumors at a secondlocation or multiple locations, e.g., in the breast.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease (e.g.,diabetes, cancer (e.g. prostate cancer, renal cancer, metastatic cancer,melanoma, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g., head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma)) means that the disease (e.g. lung cancer, ovariancancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer,kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicularcancer, leukemia, lymphoma, head and neck cancer, colorectal cancer,prostate cancer, pancreatic cancer, melanoma, breast cancer,neuroblastoma) is caused by (in whole or in part), or a symptom of thedisease is caused by (in whole or in part) the substance or substanceactivity or function.

“Patient” or “subject in need thereof” refers to a living organismsuffering from or prone to a disease or condition that can be treated byadministration of a composition or pharmaceutical composition asprovided herein. Non-limiting examples include humans, other mammals,bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and othernon-mammalian animals. In some embodiments, a patient is human.

A patient or subject for the purposes of the present invention includesboth humans and other animals, particularly mammals. Thus, the methodsare applicable to both human therapy and veterinary applications. In thepreferred embodiment the patient is a mammal, preferably a primate, andin the most preferred embodiment the patient is human.

Methods of Treating Cancer

The methods provided herein are, inter alia, useful for the treatment ofcancer. Cancer treatment can include administration of an anti-canceragent. In embodiments, an anti-cancer agent includes an adenosinereceptor antagonist, alone or in combination. In embodiments, anadenosine receptor antagonist is an A2A receptor antagonist. Throughadministration of a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist alone or in combination with a programmed celldeath protein 1 (PD-1) signaling pathway inhibitor cancer may be treatedin a subject in need thereof.

An “adenosine-A2A (A2A) receptor antagonist” as provided herein refersto a substance capable of detectably lowering expression or activitylevel of an adenosine-A2A (A2A) receptor compared to a control. Theinhibited expression or activity of the A2A receptor can be 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. Incertain instances, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, or more in comparison to a control. An “antagonist” isa compound or small molecule that inhibits an A2A receptor e.g., bybinding, partially or totally blocking stimulation, decrease, prevent,or delay activation, or inactivate, desensitize, or down-regulate signaltransduction, gene expression or enzymatic activity necessary for A2Aactivity. In embodiments, the A2A receptor antagonist is a compound or asmall molecule. In embodiments, the A2A receptor antagonist is CPI-444.In embodiments, the programmed cell death protein 1 (PD-1) signalingpathway inhibitor is atezolizumab. In embodiments, the A2A receptorantagonist and the PD-1 signaling pathway inhibitor are administeredsimultaneously or sequentially.

Likewise, a “PD-1 signaling pathway inhibitor” as provided herein refersto a substance capable of detectably lowering expression of or activitylevel of the PD-1 signaling pathway compared to a control. The inhibitedexpression or activity of the PD-1 signaling pathway can be 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. Incertain instances, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, or more in comparison to a control. An “inhibitor” is acompound or small molecule that inhibits the PD-1 signaling pathwaye.g., by binding, partially or totally blocking stimulation of the PD-1signaling pathway, decrease, prevent, or delay activation of the PD-1signaling pathway, or inactivate, desensitize, or down-regulate signaltransduction, gene expression or enzymatic activity of the PD-1signaling pathway. In embodiments, the PD-1 signaling pathway inhibitorinhibits PD-1 activity or expression. In embodiments, the PD-1 signalingpathway inhibitor inhibits PD-L1 activity or expression. In embodiments,the PD-1 signaling pathway inhibitor is a compound or a small molecule.In embodiments, the PD-1 signaling pathway inhibitor is an antibody.

According to the methods provided herein, the subject is administered aneffective amount of one or more of the agents (e.g., an A2A receptorantagonist and/or a PD-1 signaling pathway inhibitor) provided herein.An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease (e.g., cancer), reduce receptor signaling activity, reduce oneor more symptoms of a disease or condition). An example of an “effectiveamount” is an amount sufficient to contribute to the treatment,prevention, or reduction of a symptom or symptoms of a disease (e.g.,cancer), which could also be referred to as a “therapeutically effectiveamount.” A “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) means decreasing of the severity orfrequency of the symptom(s), or elimination of the symptom(s). Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. For example, for the given parameter, atherapeutically effective amount will show an increase or decrease of atleast 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least100%. In embodiments, this increase or decrease for a given parametermay vary throughout the day (e.g. a peak percentage increase or decreasemay differ from a percentage increase or decrease when therapeuticconcentrations in circulating blood are at their peak or troughconcentrations dependent on daily dosing patterns and individualpharmacokinetics). Efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over acontrol. The exact amounts will depend on the purpose of the treatment,and will be ascertainable by one skilled in the art using knowntechniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of PharmaceuticalCompounding (1999); Pickar, Dosage Calculations (1999); and Remington:The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed.,Lippincott, Williams & Wilkins).

Thus, in one aspect, a method of treating cancer in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist and a programmed cell death protein 1 (PD-1) signalingpathway inhibitor.

In embodiments, the A2A receptor antagonist is a compound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,—SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,—NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹,—C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,—SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,—NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³,—C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen, halogen, ═O,═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H,—SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In embodiments, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or submitted or unsubstituted heteroaryl.

X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I.

The symbols n₁, n₂ and n₃ are independently an integer from 0 to 4. Inembodiments, n₁ is 0. In embodiments, n₁ is 1. In embodiments, n₁ is 3.In embodiments, n₁ is 4. In embodiments, n₂ is 0. In embodiments, n₂is 1. In embodiments, n₂ is 3. In embodiments, n₂ is 4. In embodiments,n₃ is 0. In embodiments, n₃ is 1. In embodiments, n₃ is 3. Inembodiments, n₃ is 4.

The symbols m₁, m₂ and m₃ are independently an integer from 1 to 2. Inembodiments, m₁ is 0. In embodiments, m₁ is 1. In embodiments, m₁ is 2.In embodiments, m₂ is 0. In embodiments, m₂ is 1. In embodiments, m₂ is2. In embodiments, m₃ is 0. In embodiments, m₃ is 1. In embodiments, m₂is 2.

The symbols v₁, v₂ and v₃ are independently an integer from 1 to 2. Inembodiments, v₁ is 0. In embodiments, v₁ is 1. In embodiments, v₁ is 2.In embodiments, v₂ is 0. In embodiments, v₂ is 1. In embodiments, v₂ is2. In embodiments, v₃ is 0. In embodiments, v₃ is 1. In embodiments, v₃is 2.

In embodiments, R¹ is independently hydrogen, halogen, —CF₃, —CN, —CCl₃,—COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂,—NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R^(1A)-substituted or unsubstitutedalkyl, R^(1A)-substituted or unsubstituted heteroalkyl,R^(1A)-substituted or unsubstituted cycloalkyl, R^(1A)-substituted orunsubstituted heterocycloalkyl, R^(1A)-substituted or unsubstitutedaryl, or R^(1A)-substituted or unsubstituted heteroaryl. R¹ may beR^(1A)-substituted or unsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl,R^(1A)-substituted or unsubstituted (e.g., 2 to 20 membered or 2 to 6membered) heteroalkyl, R^(1A)-substituted or unsubstituted (e.g., C₃-C₈or C₅-C₇) cycloalkyl, R^(1A)-substituted or unsubstituted (e.g., 3 to 8membered or 3 to 6 membered) heterocycloalkyl, R^(1A)-substituted orunsubstituted (e.g., C₅-C₁₀ or C₅-C₆) aryl, or R^(1A)-substituted orunsubstituted (e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl.

In embodiments, R^(1A) is independently hydrogen, halogen, ═O, ═S, —CF₃,—CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H,—SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R^(1B)-substituted orunsubstituted alkyl, R^(1B)-substituted or unsubstituted heteroalkyl,R^(1B)-substituted or unsubstituted cycloalkyl, R^(1B)-substituted orunsubstituted heterocycloalkyl, R^(1B)-substituted or unsubstitutedaryl, or R^(1B)-substituted or unsubstituted heteroaryl. R^(1A) may beR^(1B)-substituted or unsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl,R^(1B)-substituted or unsubstituted (e.g., 2 to 20 membered or 2 to 6membered) heteroalkyl, R^(1B)-substituted or unsubstituted (e.g., C₃-C₈or C₅-C₇) cycloalkyl, R^(1B)-substituted or unsubstituted (e.g., 3 to 8membered or 3 to 6 membered) heterocycloalkyl, R^(1B)-substituted orunsubstituted (e.g., C₅-C₁₀ or C₅-C₆) aryl, or R^(1B)-substituted orunsubstituted (e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl.

In embodiments, R^(1B) is independently hydrogen, halogen, ═O, ═S, —CF₃,—CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H,—SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R^(1C)-substituted orunsubstituted alkyl, R^(1C)-substituted or unsubstituted heteroalkyl,R^(1C)-substituted or unsubstituted cycloalkyl, R^(1C)-substituted orunsubstituted heterocycloalkyl, R^(1C)-substituted or unsubstitutedaryl, or R^(1C)-substituted or unsubstituted heteroaryl. R^(1B) may beR^(1C)-substituted or unsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl,R^(1C)-substituted or unsubstituted (e.g., 2 to 20 membered or 2 to 6membered) heteroalkyl, R^(1C)-substituted or unsubstituted (e.g., C₃-C₈or C₅-C₇) cycloalkyl, R^(1C)-substituted or unsubstituted (e.g., 3 to 8membered or 3 to 6 membered) heterocycloalkyl, R^(1C)-substituted orunsubstituted (e.g., C₅-C₁₀ or C₅-C₆) aryl, or R^(1C)-substituted orunsubstituted (e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl.

R^(1C) is independently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃,—COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂,—NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl. R^(1C) may beindependently unsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl, unsubstituted(e.g., 2 to 20 membered or 2 to 6 membered) heteroalkyl, unsubstituted(e.g., C₃-C₈ or C₅-C₇) cycloalkyl, unsubstituted (e.g., 3 to 8 memberedor 3 to 6 membered) heterocycloalkyl, unsubstituted (e.g., C₅-C₁₀ orC₅-C₆) aryl, or unsubstituted (e.g., 5 to 10 membered or 5 to 6membered) heteroaryl.

In embodiments, R¹ is independently R^(1A)-substituted or unsubstitutedalkyl, R^(1A)-substituted or unsubstituted heteroalkyl,R^(1A)-substituted or unsubstituted cycloalkyl, R^(1A)-substituted orunsubstituted heterocycloalkyl, R^(1A)-substituted or unsubstitutedaryl, or s R^(1A)-substituted or unsubstituted heteroaryl. Inembodiments, R¹ is R^(1A)-substituted or unsubstituted (e.g., 5 to 10membered or 5 to 6 membered) heteroaryl. In embodiments, R¹ isunsubstituted 5 to 6 membered heteroaryl. In embodiments, R¹ isR^(1A)-substituted 5 to 6 membered heteroaryl. In embodiments, R¹ isunsubstituted 5 membered heteroaryl. In embodiments, R¹ isR^(1A)-substituted 5 membered heteroaryl. In embodiments, R¹ isR^(1A)-substituted furanyl.

In embodiments, R^(1A) is R^(1B)-substituted or unsubstituted (e.g.,C₁-C₂₀ or C₁-C₆) alkyl. In embodiments, R^(1A) is R^(1B)-substitutedC₁-C₆ alkyl. In embodiments, R^(1A) is unsubstituted C₁-C₆ alkyl. Inembodiments, R^(1A) is R^(1B)-substituted C₁-C₄ alkyl. In embodiments,R^(1A) is unsubstituted C₁-C₄ alkyl. In embodiments, R^(1A) isR^(1B)-substituted C₁-C₃ alkyl. In embodiments, R^(1A) is unsubstitutedC₁-C₃ alkyl. In embodiments, R^(1A) is methyl.

In embodiments, R² is independently hydrogen, halogen, —CX^(b) ₃, —CN,—SO₂Cl, —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,—NHC═(O)NR¹¹R¹², —N(O)_(m2, —NR) ¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹,—C(O)NR¹¹R¹², or —OR¹¹. In embodiments of the methods provided herein,R² is independently hydrogen, halogen, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R² is—NR¹¹R¹². In embodiments, R¹¹ and R¹² are independently hydrogen orsubstituted or unsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl. Inembodiments, R¹¹ and R¹² are independently substituted or unsubstitutedC₁-C₆ alkyl. In embodiments, R¹¹ and R¹² are independently substitutedor unsubstituted C₁-C₄ alkyl. In embodiments, R¹¹ and R¹² areindependently substituted or unsubstituted C₁-C₃ alkyl. In embodiments,R¹¹ and R¹² are independently unsubstituted C₁-C₆ alkyl. In embodiments,R¹¹ and R¹² are independently substituted or unsubstituted C₁-C₄ alkyl.In embodiments, R¹¹ and R¹² are independently unsubstituted C₁-C₃ alkyl.In embodiments, R¹¹ and R¹² are independently hydrogen.

In embodiments, R³ is independently hydrogen, halogen, —CF₃, —CN, —CCl₃,—COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂,—NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R⁴-substituted or unsubstitutedalkyl, R⁴-substituted or unsubstituted heteroalkyl, R⁴-substituted orunsubstituted cycloalkyl, R⁴-substituted or unsubstitutedheterocycloalkyl, R⁴-substituted or unsubstituted aryl, orR⁴-substituted or unsubstituted heteroaryl. R³ may be R⁴-substituted orunsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl, R⁴-substituted orunsubstituted (e.g., 2 to 20 membered or 2 to 6 membered) heteroalkyl,R⁴-substituted or unsubstituted (e.g., C₃-C₈ or C₅-C₇) cycloalkyl,R⁴-substituted or unsubstituted (e.g., 3 to 8 membered or 3 to 6membered) heterocycloalkyl, R⁴-substituted or unsubstituted (e.g.,C₅-C₁₀ or C₅-C₆) aryl, or R⁴-substituted or unsubstituted (e.g., 5 to 10membered or 5 to 6 membered) heteroaryl.

R⁴ is independently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R⁵-substituted or unsubstituted alkyl,R⁵-substituted or unsubstituted heteroalkyl, R⁵-substituted orunsubstituted cycloalkyl, R⁵-substituted or unsubstitutedheterocycloalkyl, R⁵-substituted or unsubstituted aryl, orR⁵-substituted or unsubstituted heteroaryl. R⁴ may be R⁵-substituted orunsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl, R⁵-substituted orunsubstituted (e.g., 2 to 20 membered or 2 to 6 membered) heteroalkyl,R⁵-substituted or unsubstituted (e.g., C₃-C₈ or C₅-C₇) cycloalkyl,R⁵-substituted or unsubstituted (e.g., 3 to 8 membered or 3 to 6membered) heterocycloalkyl, R⁵-substituted or unsubstituted (e.g.,C₅-C₁₀ or C₅-C₆) aryl, or R⁵-substituted or unsubstituted (e.g., 5 to 10membered or 5 to 6 membered) heteroaryl.

R⁵ is independently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R⁶-substituted or unsubstituted alkyl,R⁶-substituted or unsubstituted heteroalkyl, R⁶-substituted orunsubstituted cycloalkyl, R⁶-substituted or unsubstitutedheterocycloalkyl, R⁶-substituted or unsubstituted aryl, orR⁶-substituted or unsubstituted heteroaryl. R⁵ may be R⁶-substituted orunsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl, R⁶-substituted orunsubstituted (e.g., 2 to 20 membered or 2 to 6 membered) heteroalkyl,R⁶-substituted or unsubstituted (e.g., C₃-C₈ or C₅-C₇) cycloalkyl,R⁶-substituted or unsubstituted (e.g., 3 to 8 membered or 3 to 6membered) heterocycloalkyl, R⁶-substituted or unsubstituted (e.g.,C₅-C₁₀ or C₅-C₆) aryl, or R⁶-substituted or unsubstituted (e.g., 5 to 10membered or 5 to 6 membered) heteroaryl.

R⁶ is independently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R⁷-substituted or unsubstituted alkyl,R⁷-substituted or unsubstituted heteroalkyl, R⁷-substituted orunsubstituted cycloalkyl, R′-substituted or unsubstitutedheterocycloalkyl, R⁷-substituted or unsubstituted aryl, orR⁷-substituted or unsubstituted heteroaryl. R⁶ may be R⁷-substituted orunsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl, R⁷-substituted orunsubstituted (e.g., 2 to 20 membered or 2 to 6 membered) heteroalkyl,R⁷-substituted or unsubstituted (e.g., C₃-C₈ or C₅-C₇) cycloalkyl,R⁷-substituted or unsubstituted (e.g., 3 to 8 membered or 3 to 6membered) heterocycloalkyl, R⁷-substituted or unsubstituted (e.g.,C₅-C₁₀ or C₅-C₆) aryl, or R⁷-substituted or unsubstituted (e.g., 5 to 10membered or 5 to 6 membered) heteroaryl.

In embodiments, R³ is independently hydrogen, halogen, R⁴-substituted orunsubstituted alkyl, R⁴-substituted or unsubstituted heteroalkyl,R⁴-substituted or unsubstituted cycloalkyl, R⁴-substituted orunsubstituted heterocycloalkyl, R⁴-substituted or unsubstituted aryl, orR⁴-substituted or unsubstituted heteroaryl. In embodiments, R³ isindependently R⁴-substituted or unsubstituted (e.g., C₁-C₂₀ or C₁-C₆)alkyl. In embodiments, R³ is independently R⁴-substituted orunsubstituted C₁-C₆ alkyl. In embodiments, R³ is independentlyR⁴-substituted or unsubstituted C₁-C₅ alkyl. In embodiments, R³ isindependently R⁴-substituted or unsubstituted C₁-C₄ alkyl. Inembodiments, R³ is independently R⁴-substituted or unsubstituted C₁-C₃alkyl. In embodiments, R³ is independently unsubstituted C₁-C₆ alkyl. Inembodiments, R³ is independently unsubstituted C₁-C₅ alkyl. Inembodiments, R³ is independently R⁴-unsubstituted C₁-C₄ alkyl. Inembodiments, R³ is independently unsubstituted C₁-C₃ alkyl. Inembodiments, R³ is independently R⁴-substituted C₁-C₆ alkyl. Inembodiments, R³ is independently R⁴-substituted C₁-C₅ alkyl. Inembodiments, R³ is independently R⁴-substituted C₁-C₄ alkyl. Inembodiments, R³ is independently R⁴-substituted C₁-C₃ alkyl. Inembodiments, R³ is R⁴-substituted C₁ alkyl.

In embodiments, R⁴ is R⁵-substituted or unsubstituted (e.g., C₁-C₂₀ orC₁-C₆) alkyl, R⁵-substituted or unsubstituted (e.g., 2 to 20 membered or2 to 6 membered) heteroalkyl, R⁵-substituted or unsubstituted (e.g.,C₃-C₈ or C₅-C₇) cycloalkyl, R⁵-substituted or unsubstituted (e.g., 3 to8 membered or 3 to 6 membered) heterocycloalkyl, R⁵-substituted orunsubstituted (e.g., C₅-C₁₀ or C₅-C₆) aryl, or R⁵-substituted orunsubstituted (e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl. Inembodiments, R⁴ is R⁵-substituted or unsubstituted 5 to 6 memberedheteroaryl. In embodiments, R⁴ is R⁵-substituted or unsubstituted 6membered heteroaryl. In embodiments, R⁴ is unsubstituted 6 memberedheteroaryl. In embodiments, R⁴ is R⁵-substituted 6 membered heteroaryl.In embodiments, R⁴ is R⁵-substituted pyridinyl.

In embodiments, R⁵ is R⁶-substituted or unsubstituted (e.g., C₁-C₂₀ orC₁-C₆) alkyl, R⁶-substituted or unsubstituted (e.g., 2 to 20 membered or2 to 6 membered) heteroalkyl, R⁶-substituted or unsubstituted (e.g.,C₃-C₈ or C₅-C₇) cycloalkyl, R⁶-substituted or unsubstituted (e.g., 3 to8 membered or 3 to 6 membered) heterocycloalkyl, R⁶-substituted orunsubstituted (e.g., C₅-C₁₀ or C₅-C₆) aryl, or R⁶-substituted orunsubstituted (e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl. Inembodiments, R⁵ is R⁶-substituted or unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R⁵ is R⁶-substituted or unsubstituted 2 to5 membered heteroalkyl. In embodiments, R⁵ is R⁶-substituted orunsubstituted 2 to 4 membered heteroalkyl. In embodiments, R⁵ isR⁶-substituted or unsubstituted 2 to 3 membered heteroalkyl. Inembodiments, R⁵ is R⁶-substituted or unsubstituted 2 memberedheteroalkyl. In embodiments, R⁵ is unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R⁵ is unsubstituted 2 to 5 memberedheteroalkyl. In embodiments, R⁵ is unsubstituted 2 to 4 memberedheteroalkyl. In embodiments, R⁵ unsubstituted 2 to 3 memberedheteroalkyl. In embodiments, R⁵ is unsubstituted 2 membered heteroalkyl.In embodiments, R⁵ is R⁶-substituted 2 to 6 membered heteroalkyl. Inembodiments, R⁵ is R⁶-substituted 2 to 5 membered heteroalkyl. Inembodiments, R⁵ is R⁶-substituted 2 to 4 membered heteroalkyl. Inembodiments, R⁵ is R⁶-substituted 2 to 3 membered heteroalkyl. Inembodiments, R⁵ is R⁶-substituted 2 membered heteroalkyl.

In embodiments, R⁶ is R⁷-substituted or unsubstituted (e.g., C₁-C₂₀ orC₁-C₆) alkyl, R⁷-substituted or unsubstituted (e.g., 2 to 20 membered or2 to 6 membered) heteroalkyl, R⁷-substituted or unsubstituted (e.g.,C₃-C₈ or C₅-C₇) cycloalkyl, R⁷-substituted or unsubstituted (e.g., 3 to8 membered or 3 to 6 membered) heterocycloalkyl, R⁷-substituted orunsubstituted (e.g., C₅-C₁₀ or C₅-C₆) aryl, or R⁷-substituted orunsubstituted (e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl. Inembodiments, R⁶ is R⁷-substituted or unsubstituted 3 to 6 memberedheterocycloalkyl. In embodiments, R⁶ is R⁷-substituted or unsubstituted5 membered heterocycloalkyl. In embodiments, R⁶ is R⁷-substituted 5membered heterocycloalkyl. In embodiments, R⁶ is unsubstituted 5membered heterocycloalkyl. In embodiments, R⁶ is unsubstitutedtetrahydrofuranyl.

In embodiments of the methods provided herein, R⁹, R¹⁰, R¹¹, R¹², R¹³and R¹⁴ are independently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃,—COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂,—NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl.

In embodiments, R¹ is R^(1A)-substituted furanyl. In one furtherembodiment, R^(1A) is methyl. In another further embodiment, R² is—NR¹¹R¹². In another further embodiment, R¹¹ and R¹² are independentlyhydrogen. In yet another further embodiment, R³ is R⁴-substituted C₁alkyl. In another further embodiment, R⁴ is R⁵-substituted pyridinyl. Inyet another further embodiment, R⁵ is R⁶-substituted 2 memberedheteroalkyl. In another further embodiments, R⁶ is unsubstitutedtetrahydrofuranyl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In embodiments, R⁶, R^(6.1) andR^(6.2) are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, R^(6.1) and R^(6.2) are hydrogen and R6 is a substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In embodiments, R^(6.1) and R^(6.2) arehydrogen and R6 is substituted or unsubstituted heterocycloalkyl. Inembodiments, R^(6.1) and R^(6.2) are hydrogen and R6 is unsubstitutedheterocycloalkyl. In embodiments, R¹ is substituted (e.g. with anunsubstituted C₁-C₅ alkyl) or unsubstituted heteroaryl. In embodiments,R¹ is substituted (e.g. with an unsubstituted C₁-C₅ alkyl) orunsubstituted furanyl. In embodiments, R¹ is methyl-substituted furanyl.

In formula (II), R¹ and R⁶ are as described above (e.g., R⁶ may beR⁷-substituted or unsubstituted 3 to 6 membered heterocycloalkyl and R¹may be R^(1A)-substituted 5 to 6 membered heteroaryl). Thus, inembodiments, R⁶ is unsubstituted tetrahydrofuranyl and R¹ isR^(1A)-substituted furanyl.

In formula (II), R^(6.1) may be independently hydrogen, halogen, —CF₃,—CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H,—SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R^(7.1)-substitutedor unsubstituted alkyl, R^(7.1)-substituted or unsubstitutedheteroalkyl, R^(7.1)-substituted or unsubstituted cycloalkyl,R^(7.1)-substituted or unsubstituted heterocycloalkyl,R^(7.1)-substituted or unsubstituted aryl, or R^(7.1)-substituted orunsubstituted heteroaryl. R^(6.1) may be R^(7.1)-substituted orunsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl, R^(7.1)-substituted orunsubstituted (e.g., 2 to 20 membered or 2 to 6 membered) heteroalkyl,R^(7.1)-substituted or unsubstituted (e.g., C₃-C₈ or C₅-C₇) cycloalkyl,R^(7.1)-substituted or unsubstituted (e.g., 3 to 8 membered or 3 to 6membered) heterocycloalkyl, R^(7.1)-substituted or unsubstituted (e.g.,C₅-C₁₀ or C₅-C₆) aryl, or R^(7.1)-substituted or unsubstituted (e.g., 5to 10 membered or 5 to 6 membered) heteroaryl. In embodiments, R^(6.1)is R^(7.1)-substituted or unsubstituted C₁-C₆ alkyl. In embodiments,R^(6.1) is R^(7.1)-substituted or unsubstituted C₁-C₅ alkyl. Inembodiments, R^(6.1) is R^(7.1)-substituted or unsubstituted C₁-C₄alkyl. In embodiments, R^(6.1) is R^(7.1)-substituted or unsubstitutedC₁-C₃ alkyl. In embodiments, R^(6.1) is R^(7.1)-substituted C₁-C₆ alkyl.In embodiments, R^(6.1) is R^(7.1)-substituted C₁-C₅ alkyl. Inembodiments, R^(6.1) is R^(7.1)-substituted C₁-C₄ alkyl. In embodiments,R^(6.1) is R^(7.1)-substituted C₁-C₃ alkyl. In embodiments, R^(6.1) isunsubstituted C₁-C₆ alkyl. In embodiments, R^(6.1) is unsubstitutedC₁-C₅ alkyl. In embodiments, R^(6.1) is unsubstituted C₁-C₄ alkyl. Inembodiments, R^(6.1) is unsubstituted C₁-C₃ alkyl. In embodiments,R^(6.1) is unsubstituted methyl.

R^(6.2) is independently hydrogen, halogen, ═O, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, R^(7.2)-substituted or unsubstitutedalkyl, R^(7.2)-substituted or unsubstituted heteroalkyl,R^(7.2)-substituted or unsubstituted cycloalkyl, R^(7.2)-substituted orunsubstituted heterocycloalkyl, R^(7.2)-substituted or unsubstitutedaryl, or R^(7.2)-substituted or unsubstituted heteroaryl. R^(6.2) may beR^(7.2)-substituted or unsubstituted (e.g., C₁-C₂₀ or C₁-C₆) alkyl,R^(7.2)-substituted or unsubstituted (e.g., 2 to 20 membered or 2 to 6membered) heteroalkyl, R^(7.2)-substituted or unsubstituted (e.g., C₃-C₈or C₅-C₇) cycloalkyl, R^(7.2)-substituted or unsubstituted (e.g., 3 to 8membered or 3 to 6 membered) heterocycloalkyl, R^(7.2)-substituted orunsubstituted (e.g., C₅-C₁₀ or C₅-C₆) aryl, or R^(7.2)-substituted orunsubstituted (e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl. Inembodiments, R^(6.2) is R^(7.2)-substituted or unsubstituted C₁-C₆alkyl. In embodiments, R^(6.2) is R^(7.2)-substituted or unsubstitutedC₁-C₅ alkyl. In embodiments, R^(6.2) is substituted or unsubstitutedC₁-C₄ alkyl. In embodiments, R^(6.2) is R^(7.2)-substituted orunsubstituted C₁-C₃ alkyl. In embodiments, R^(6.2) isR^(7.2)-substituted C₁-C₆ alkyl. In embodiments, R^(6.2) is substitutedC₁-C₅ alkyl. In embodiments, R^(6.2) is R^(7.2)-substituted C₁-C₄ alkyl.In embodiments, R^(6.2) is R^(7.2)-substituted C₁-C₃ alkyl. Inembodiments, R^(6.2) is unsubstituted C₁-C₆ alkyl. In embodiments,R^(6.2) is unsubstituted C₁-C₅ alkyl. In embodiments, R^(6.2) isunsubstituted C₁-C₄ alkyl. In embodiments, R^(6.2) is unsubstitutedC₁-C₃ alkyl. In embodiments, R^(6.2) is unsubstituted methyl.

R⁷, R^(7.1) and R^(7.2) are independently hydrogen, halogen, ═O, ═S,—CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H,—SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl. R⁷, R^(7.1) and R^(7.2) may be independently unsubstituted(e.g., C₁-C₂₀ or C₁-C₆) alkyl, unsubstituted (e.g., 2 to 20 membered or2 to 6 membered) heteroalkyl, unsubstituted (e.g., C₃-C₈ or C₅-C₇)cycloalkyl, unsubstituted (e.g., 3 to 8 membered or 3 to 6 membered)heterocycloalkyl, unsubstituted (e.g., C₅-C₁₀ or C₅-C₆) aryl, orunsubstituted (e.g., 5 to 10 membered or 5 to 6 membered) heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

All compounds provided herein may optionally be provided as apharmaceutically acceptable salt.

In embodiments, the PD-1 signaling pathway inhibitor is a programmeddeath-ligand 1 (PD-L1) antagonist or a PD-1 antagonist. A PD-L1antagonist as provided herein is a substance that, at least in part,partially or totally blocks stimulation, decreases, prevents, or delaysactivation, or inactivates, desensitizes, or down-regulates signaltransduction of PD-L1. Likewise, a PD-1 antagonist as provided herein isa substance that, at least in part, partially or totally blocksstimulation, decreases, prevents, or delays activation, or inactivates,desensitizes, or down-regulates signal transduction of PD-1. Inembodiments, the programmed death-ligand 1 (PD-L1) antagonist is anantibody or a small molecule. In embodiments, the PD-L1 antagonist is anantibody. In embodiments, the antibody is atezolizumab. The term“atezolizumab” refers to a fully humanized, engineered monoclonalantibody of IgG1 isotype against the protein programmed cell deathligand 1 (PD-L1). Atezolizumab is also known as “MPDL3280A.” In thecustomary sense, atezolizumab refers to CAS Registry number1380723-44-3.

In embodiments, the PD-1 antagonist is an antibody or a small molecule.

In embodiments, an adenosine receptor antagonists is administration inconjunction with an additional anti-cancer agent. In embodiments, anadenosine receptor antagonist is administered in conjunction with anantibody anti-cancer agent. In embodiments, an adenosine receptorantagonist is administered with a PD-L1 antagonist. In embodiments, anA2A receptor antagonist is administered in conjunction with an antibodyagainst PD-L1. In embodiments, CPI-444 is administered in conjunctionwith azetolizumab.

In embodiments, the A2A receptor antagonist and the PD-1 signalingpathway inhibitor are administered in a combined synergistic amount. A“combined synergistic amount” as used herein refers to the sum of afirst amount (e.g., an amount of an A2A receptor antagonist) and asecond amount (e.g., an amount of a PD-1 signaling pathway inhibitor)that results in a synergistic effect (i.e. an effect greater than anadditive effect). Therefore, the terms “synergy”, “synergism”,“synergistic”, “combined synergistic amount”, and “synergistictherapeutic effect” which are used herein interchangeably, refer to ameasured effect of compounds administered in combination where themeasured effect is greater than the sum of the individual effects ofeach of the compounds administered alone as a single agent.

In embodiments, a synergistic amount may be about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the amountof the A2A receptor antagonist when used separately from the PD-1signaling pathway inhibitor. In embodiments, a synergistic amount may beabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3,8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% of the amount of the PD-1 signaling pathway inhibitorwhen used separately from the A2A receptor antagonist.

The synergistic effect may be an A2A receptor activity decreasing effectand/or a PD-1 signaling pathway activity decreasing effect. Inembodiments, synergy between the A2A receptor antagonist and the PD-1signaling pathway inhibitor may result in about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% greaterdecrease (e.g., decrease of A2A receptor activity or decrease of PD-1signaling pathway activity) than the sum of the decrease of the A2Areceptor antagonist or the PD-1 signaling pathway when used individuallyand separately. In embodiments, synergy between the A2A receptorantagonist and the PD-1 signaling pathway inhibitor may result in 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100% greater inhibition of the A2A receptor and/or the PD-1signaling pathway than the sum of the inhibition of the A2A receptorantagonist or the PD-1 signaling pathway inhibitor when usedindividually and separately.

The synergistic effect may be a cancer-treating effect such as an lungcancer (i.e. a lung cancer-treating synergistic effect), bladder cancer(i.e. a bladder cancer-treating synergistic effect), melanoma (i.e. amelanoma-treating synergistic effect), renal cell carcinoma (i.e. arenal cell carcinoma-treating synergistic effect), colon cancer (i.e. acolon cancer-treating synergistic effect), ovarian cancer (i.e. anovarian cancer-treating synergistic effect), gastric cancer (i.e. agastric cancer-treating synergistic effect), breast cancer (i.e. abreast cancer-treating synergistic effect), head and neck carcinoma(i.e. a head and neck carcinoma-treating synergistic effect), prostatecancer (i.e. a prostate cancer-treating synergistic effect) and ahematologic malignancy (i.e. a hematologic malignancy-treatingsynergistic effect).

The A2A receptor antagonist and the PD-1 signaling pathway inhibitor maybe administered in combination either concomitantly (e.g., as amixture), separately but simultaneously (e.g., via separate intravenouslines) or sequentially (e.g., one agent is administered first followedby administration of the second agent). Thus, the term combination isused to refer to concomitant, simultaneous or sequential administrationof the A2A receptor antagonist and the PD-1 signaling pathway inhibitor.In embodiments, where the A2A receptor antagonist and the PD-1 signalingpathway inhibitor are administered sequentially, the A2A receptorantagonist is administered at a first time point and the PD-1 signalingpathway inhibitor is administered at a second time point, wherein thefirst time point precedes the second time point. The course of treatmentis best determined on an individual basis depending on the particularcharacteristics of the subject and the type of treatment selected. Thetreatment, such as those disclosed herein, can be administered to thesubject on a daily, twice daily, bi-weekly, monthly or any applicablebasis that is therapeutically effective. The treatment can beadministered alone or in combination with any other treatment disclosedherein or known in the art. The additional treatment can be administeredsimultaneously with the first treatment, at a different time, or on anentirely different therapeutic schedule (e.g., the first treatment canbe daily, while the additional treatment is weekly). Thus, inembodiments, the A2A receptor antagonist and the PD-1 signaling pathwayinhibitor are administered simultaneously or sequentially.

In embodiments, the A2A receptor antagonist is administered at a firsttime point and the PD-1 signaling pathway inhibitor is administered at asecond time point, wherein the first time point precedes the second timepoint. In embodiments, the second time point is within less than about120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 11, 9,8, 7, 6, 5, 4, 3, 2 or 1 days from the first time point. In embodiments,the second time point is within less than about 120 days from the firsttime point. In embodiments, the second time point is within less thanabout 90 days from the first time point. In embodiments, the second timepoint is within less than about 60 days from the first time point. Inembodiments, the second time point is within less than about 50 daysfrom the first time point. In embodiments, the second time point iswithin less than about 40 days from the first time point. Inembodiments, the second time point is within less than about 30 daysfrom the first time point. In embodiments, the second time point iswithin less than about 20 days from the first time point.

In embodiments, the second time point is within less than about 19 daysfrom the first time point. In embodiments, the second time point iswithin less than about 18 days from the first time point. Inembodiments, the second time point is within less than about 17 daysfrom the first time point. In embodiments, the second time point iswithin less than about 16 days from the first time point. Inembodiments, the second time point is within less than about 15 daysfrom the first time point. In embodiments, the second time point iswithin less than about 14 days from the first time point. Inembodiments, the second time point is within less than about 13 daysfrom the first time point. In embodiments, the second time point iswithin less than about 12 days from the first time point. Inembodiments, the second time point is within less than about 11 daysfrom the first time point. In embodiments, the second time point iswithin less than about 10 days from the first time point. Inembodiments, the second time point is within less than about 9 days fromthe first time point. In embodiments, the second time point is withinless than about 8 days from the first time point. In embodiments, thesecond time point is within less than about 7 days from the first timepoint. In embodiments, the second time point is within less than about 6days from the first time point. In embodiments, the second time point iswithin less than about 5 days from the first time point. In embodiments,the second time point is within less than about 4 days from the firsttime point. In embodiments, the second time point is within less thanabout 3 days from the first time point. In embodiments, the second timepoint is within less than about 2 days from the first time point. Inembodiments, the second time point is within less than about 1 day fromthe first time point.

In embodiments, the second time point is within about 8, 10 or 12 daysfrom the first time point. In embodiments, the second time point iswithin about 8, days from the first time point. In embodiments, thesecond time point is within about 10 days from the first time point. Inembodiments, the second time point is within about 12 days from thefirst time point. In embodiments, the PD-1 signaling pathway inhibitorand the A2A receptor antagonist are simultaneously administered at thesecond time point. In embodiments, the PD-1 signaling pathway inhibitorand the A2A receptor antagonist are concomitantly administered at thesecond time point. In embodiments, the PD-1 signaling pathway inhibitoris administered at the second time point and the A2A receptor antagonistis not administered at the second time point.

In embodiments, the PD-1 signaling pathway inhibitor is administered ata first time point and the A2A receptor antagonist is administered at asecond time point, wherein the first time point precedes the second timepoint. In embodiments, the second time point is within less than about120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 11, 9,8, 7, 6, 5, 4, 3, 2 or 1 days from the first time point. In embodiments,the second time point is within less than about 120 days from the firsttime point. In embodiments, the second time point is within less thanabout 90 days from the first time point. In embodiments, the second timepoint is within less than about 60 days from the first time point. Inembodiments, the second time point is within less than about 50 daysfrom the first time point. In embodiments, the second time point iswithin less than about 40 days from the first time point. Inembodiments, the second time point is within less than about 30 daysfrom the first time point. In embodiments, the second time point iswithin less than about 20 days from the first time point.

In embodiments, the second time point is within less than about 19 daysfrom the first time point. In embodiments, the second time point iswithin less than about 18 days from the first time point. Inembodiments, the second time point is within less than about 17 daysfrom the first time point. In embodiments, the second time point iswithin less than about 16 days from the first time point. Inembodiments, the second time point is within less than about 15 daysfrom the first time point. In embodiments, the second time point iswithin less than about 14 days from the first time point. Inembodiments, the second time point is within less than about 13 daysfrom the first time point. In embodiments, the second time point iswithin less than about 12 days from the first time point. Inembodiments, the second time point is within less than about 11 daysfrom the first time point. In embodiments, the second time point iswithin less than about 10 days from the first time point. Inembodiments, the second time point is within less than about 9 days fromthe first time point. In embodiments, the second time point is withinless than about 8 days from the first time point. In embodiments, thesecond time point is within less than about 7 days from the first timepoint. In embodiments, the second time point is within less than about 6days from the first time point. In embodiments, the second time point iswithin less than about 5 days from the first time point. In embodiments,the second time point is within less than about 4 days from the firsttime point. In embodiments, the second time point is within less thanabout 3 days from the first time point. In embodiments, the second timepoint is within less than about 2 days from the first time point. Inembodiments, the second time point is within less than about 1 day fromthe first time point.

In embodiments, the second time point is within about 8, 10 or 12 daysfrom the first time point. In embodiments, the second time point iswithin about 8, days from the first time point. In embodiments, thesecond time point is within about 10 days from the first time point. Inembodiments, the second time point is within about 12 days from thefirst time point. In embodiments, the PD-1 signaling pathway inhibitorand the A2A receptor antagonist are simultaneously administered at thesecond time point. In embodiments, the PD-1 signaling pathway inhibitorand the A2A receptor antagonist are concomitantly administered at thesecond time point. In embodiments, the A2A receptor antagonist isadministered at the second time point and the PD-1 signaling pathwayinhibitor is not administered at the second time point.

According to the methods provided herein, the subject is administered aneffective amount of one or more of the agents (e.g., an A2A receptorantagonist and/or a PD-1 signaling pathway inhibitor) provided herein.An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease (e.g., cancer), reduce receptor signalling activity, reduce oneor more symptoms of a disease or condition). An example of an “effectiveamount” is an amount sufficient to contribute to the treatment,prevention, or reduction of a symptom or symptoms of a disease (e.g.,cancer), which could also be referred to as a “therapeutically effectiveamount.” A “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) means decreasing of the severity orfrequency of the symptom(s), or elimination of the symptom(s). Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. For example, for the given parameter, atherapeutically effective amount will show an increase or decrease of atleast 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least100%. Efficacy can also be expressed as “-fold” increase or decrease.For example, a therapeutically effective amount can have at least a1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. Theexact amounts will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see,e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd,The Art, Science and Technology of Pharmaceutical Compounding (1999);Pickar, Dosage Calculations (1999); and Remington: The Science andPractice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott,Williams & Wilkins).

In embodiments, the A2A receptor antagonist is administered at an amountof about 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg or 300 mg/kg. In embodiments, theA2A receptor antagonist is administered at an amount of about 0.5 mg/kg.In embodiments, the A2A receptor antagonist is administered at an amountof about 1 mg/kg. In embodiments, the A2A receptor antagonist isadministered at an amount of about 5 mg/kg. In embodiments, the A2Areceptor antagonist is administered at an amount of about 10 mg/kg. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 20 mg/kg. In embodiments, the A2A receptor antagonist isadministered at an amount of about 30 mg/kg. In embodiments, the A2Areceptor antagonist is administered at an amount of about 40 mg/kg. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 50 mg/kg. In embodiments, the A2A receptor antagonist isadministered at an amount of about 60 mg/kg. In embodiments, the A2Areceptor antagonist is administered at an amount of about 70 mg/kg. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 80 mg/kg. In embodiments, the A2A receptor antagonist isadministered at an amount of about 90 mg/kg. In embodiments, the A2Areceptor antagonist is administered at an amount of about 100 mg/kg. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 200 mg/kg. In embodiments, the A2A receptor antagonist isadministered at an amount of about 300 mg/kg. It is understood thatwhere the amount is referred to as “mg/kg”, the amount is milligram perkilogram body weight of the subject being administered with the A2Areceptor antagonist.

In embodiments, the A2A receptor antagonist is administered at an amountof about 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200mg/kg or 300 mg/kg. In embodiments, the A2A receptor antagonist isadministered at an amount of about 1 mg/kg. In embodiments, the A2Areceptor antagonist is administered at an amount of about 1 mg/kg to 2mg/kg. In embodiments, the A2A receptor antagonist is administered at anamount of about 1 mg/kg to 3 mg/kg. In embodiments, the A2A receptorantagonist is administered at an amount of about 1 mg/kg to 4 mg/kg. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 1 mg/kg to 5 mg/kg.

In embodiments, the A2A receptor antagonist is administered at an amountof about 10 mg BID, 20 mg BID, 30 mg BID, 40 mg BID, 50 mg BID, 60 mgBID, 70 mg BID, 80 mg BID, 90 mg BID, 100 mg BID, 110 mg BID, 120 mgBID, 130 mg BID, 140 mg BID, 150 mg BID, 160 mg BID, 170 mg BID, 180 mgBID, 190 mg BID, 200 mg BID, 210 mg BID, 220 mg BID, 230 mg BID, 240 mgBID, 250 mg BID, 260 mg BID, 270 mg BID, 280 mg BID, 290 mg BID, or 300mg BID. In embodiments, the A2A receptor antagonist is administered atan amount of about 10 mg BID. In embodiments, the A2A receptorantagonist is administered at an amount of about 20 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 30 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 40 mg BID. In embodiments, the A2Areceptor antagonist is administered at an amount of about 50 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 60 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 70 mg BID. In embodiments, the A2Areceptor antagonist is administered at an amount of about 80 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 90 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 100 mg BID. It is understood thatwhere the amount is referred to as “BID” which stands for “bis in die”,the amount is administered twice a day.

In embodiments, the A2A receptor antagonist is administered at an amountof about 110 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 120 mg BID. In embodiments, the A2Areceptor antagonist is administered at an amount of about 130 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 140 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 150 mg BID. In embodiments, the A2Areceptor antagonist is administered at an amount of about 160 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 170 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 180 mg BID. In embodiments, the A2Areceptor antagonist is administered at an amount of about 190 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 200 mg BID. It is understood that where the amount is referred toas “BID” which stands for “bis in die”, the amount is administered twicea day.

In embodiments, the A2A receptor antagonist is administered at an amountof about 210 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 220 mg BID. In embodiments, the A2Areceptor antagonist is administered at an amount of about 230 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 240 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 250 mg BID. In embodiments, the A2Areceptor antagonist is administered at an amount of about 260 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 270 mg BID. In embodiments, the A2A receptor antagonist isadministered at an amount of about 280 mg BID. In embodiments, the A2Areceptor antagonist is administered at an amount of about 290 mg BID. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 300 mg BID. It is understood that where the amount is referred toas “BID” which stands for “bis in die”, the amount is administered twicea day.

In embodiments, the A2A receptor antagonist is administered at an amountof about 10 mg QD, 20 mg QD, 30 mg QD, 40 mg QD, 50 mg QD, 60 mg QD, 70mg QD, 80 mg QD, 90 mg QD, 100 mg QD, 110 mg QD, 120 mg QD, 130 mg QD,140 mg QD, 150 mg QD, 160 mg QD, 170 mg QD, 180 mg QD, 190 mg QD, 200 mgQD, 210 mg QD, 220 mg QD, 230 mg QD, 240 mg QD, 250 mg QD, 260 mg QD,270 mg QD, 280 mg QD, 290 mg QD, or 300 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 10 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 20 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 30 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 40 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 50 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 60 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 70 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 80 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 90 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 100 mg QD. Itis understood that where the amount is referred to as “QD” which standsfor “quaque die”, the amount is administered once a day.

In embodiments, the A2A receptor antagonist is administered at an amountof about 110 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 120 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 130 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 140 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 150 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 160 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 170 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 180 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 190 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 200 mg QD. It is understood that where the amount is referred toas “QD” which stands for “quaque die”, the amount is administered once aday.

In embodiments, the A2A receptor antagonist is administered at an amountof about 210 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 220 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 230 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 240 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 250 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 260 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 270 mg QD. In embodiments, the A2A receptor antagonist isadministered at an amount of about 280 mg QD. In embodiments, the A2Areceptor antagonist is administered at an amount of about 290 mg QD. Inembodiments, the A2A receptor antagonist is administered at an amount ofabout 300 mg QD. It is understood that where the amount is referred toas “QD” which stands for “quaque die”, the amount is administered once aday.

The A2A receptor antagonist may be administered at an amount as providedherein on 28 consecutive days. The A2A receptor antagonist may beadministered at an amount as provided herein on 14 consecutive days. Inembodiments, the A2A receptor antagonist is administered at 50 mg BID,100 mg BID or 200 mg QD. In embodiments, the A2A receptor antagonist isadministered at 50 mg BID. In embodiments, the A2A receptor antagonistis administered at 100 mg BID. In embodiments, the A2A receptorantagonist is administered at 200 mg QD. In embodiments, the A2Areceptor antagonist is administered at 100 mg BID and the PD-1 signalingpathway inhibitor is administered at an amount of 840 mg. In furtherembodiments, the A2A receptor antagonist and the PD-1 signaling pathwayinhibitor are administered simultaneously on 28 consecutive days. Inother further embodiments, the A2A receptor antagonist and the PD-1signaling pathway inhibitor are administered simultaneously on 14consecutive days.

In embodiments, the PD-1 signaling pathway inhibitor is administered atan amount of less than about 1,300 mg. In embodiments, the PD-1signaling pathway inhibitor is administered at an amount of less thanabout 1,200 mg. In embodiments, the PD-1 signaling pathway inhibitor isadministered at an amount of less than about 1,100 mg. In embodiments,the PD-1 signaling pathway inhibitor is administered at an amount ofless than about 1,000 mg. In embodiments, the PD-1 signaling pathwayinhibitor is administered at an amount of less than about 900 mg. Inembodiments, the PD-1 signaling pathway inhibitor is administered at anamount of less than about 800 mg. In embodiments, the PD-1 signalingpathway inhibitor is administered at an amount of less than about 700mg. In embodiments, the PD-1 signaling pathway inhibitor is administeredat an amount of less than about 600 mg. In embodiments, the PD-1signaling pathway inhibitor is administered at an amount of less thanabout 500 mg. In embodiments, the PD-1 signaling pathway inhibitor isadministered at an amount of less than about 400 mg. In embodiments, thePD-1 signaling pathway inhibitor is administered at an amount of lessthan about 300 mg. In embodiments, the PD-1 signaling pathway inhibitoris administered at an amount of less than about 200 mg. In embodiments,the PD-1 signaling pathway inhibitor is administered at an amount ofless than about 100 mg. In embodiments, the PD-1 signaling pathwayinhibitor is administered at an amount of about 100 mg, 200 mg, 300 mg,400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1.00 mg, 1,100 mg, 1,200mg, or 1,300 mg. It is understood that where the amount is referred toas “mg” that the amount is the total amount in milligram of PD-1signaling pathway inhibitor administered to the subject.

In embodiments, the PD-1 signaling pathway inhibitor is administered atan amount of about 700 mg. In embodiments, the PD-1 signaling pathwayinhibitor is administered at an amount of about 720 mg. In embodiments,the PD-1 signaling pathway inhibitor is administered at an amount ofabout 740 mg. In embodiments, the PD-1 signaling pathway inhibitor isadministered at an amount of about 760 mg. In embodiments, the PD-1signaling pathway inhibitor is administered at an amount of about 780mg. In embodiments, the PD-1 signaling pathway inhibitor is administeredat an amount of about 800 mg. In embodiments, the PD-1 signaling pathwayinhibitor is administered at an amount of about 820 mg. In embodiments,the PD-1 signaling pathway inhibitor is administered at an amount ofabout 840 mg. In embodiments, the PD-1 signaling pathway inhibitor isadministered at an amount of about 860 mg. In embodiments, the PD-1signaling pathway inhibitor is administered at an amount of about 880mg. In embodiments, the PD-1 signaling pathway inhibitor is administeredat an amount of about 900 mg. It is understood that where the amount isreferred to as “mg” that the amount is the total amount in milligram ofPD-1 signaling pathway inhibitor administered to the subject.

The methods provided herein are, inter alia, useful for the treatment ofcancer. In embodiments, the cancer is selected from lung cancer, bladdercancer, melanoma, renal cell carcinoma, colon cancer, ovarian cancer,gastric cancer, breast cancer, head and neck carcinoma, prostate cancerand a hematologic malignancy. In embodiments, the cancer is lung cancer.In embodiments, the cancer is bladder cancer. In embodiments, the canceris melanoma. In embodiments, the cancer is renal cell carcinoma. Inembodiments, the cancer is colon cancer. In embodiments, the cancer isovarian cancer. In embodiments, the cancer is gastric cancer. Inembodiments, the cancer is breast cancer. In embodiments, the cancer ishead and neck carcinoma. In embodiments, the cancer is prostate cancer.In embodiments, the cancer is a hematologic malignancy.

In another aspect, a method of treating cancer in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,—SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,—NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹,—C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,—SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,—NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³,—C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen, halogen, ═O,═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H,—SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I.

n₁, n₂ and n₃ are independently an integer from 0 to 4.

m₁, m₂ and m₃ are independently an integer from 1 to 2.

And v₁, v₂ and v₃ are independently an integer from 1 to 2.

The A2A receptor antagonist provided herein is the same A2A receptorantagonist as described above for aspects of treating cancer using anA2A receptor antagonist and a PD-1 signaling pathway inhibitor.Therefore, the definitions for substituents and variables of formula (I)and (II) are the same as described above (e.g., R¹ is R^(1A)-substitutedfuranyl; R^(1A) is methyl; R² is —NR¹¹R¹²; R¹¹ and R¹² are independentlyhydrogen; R³ is R⁴-substituted C₁ alkyl; R⁴ is R⁵-substituted pyridinyl;R⁵ is R⁶-substituted 2 membered heteroalkyl; R⁶ is unsubstitutedtetrahydrofuranyl and are incorporated herewith.

Thus, in embodiments, the A2A receptor antagonist is a compound offormula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the method further includes administering atherapeutically effective amount of a PD-1 signaling pathway inhibitor.In embodiments, the A2A receptor antagonist and the PD-1 signalingpathway inhibitor are administered in a combined synergistic amount. Inembodiments, the A2A receptor antagonist and the PD-1 signaling pathwayinhibitor are administered simultaneously or sequentially. Inembodiments, the A2A receptor antagonist is administered at a first timepoint and the PD-1 signaling pathway inhibitor is administered at asecond time point, wherein the first time point precedes the second timepoint. In embodiments, the second time point is within less than about120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 11, 9,8, 7, 6, 5, 4, 3, 2, or 1 days from the first time point. Inembodiments, the second time point is within about 8, 10 or 12 days fromthe first time point. In embodiments, the PD-1 signaling pathwayinhibitor is administered at a first time point and the A2A receptorantagonist is administered at a second time point, wherein the firsttime point precedes the second time point. In embodiments, the secondtime point is within less than about 120, 90, 60, 50, 40, 30, 20, 19,18, 17, 16, 15, 14, 13, 12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 daysfrom the first time point. In embodiments, the second time point iswithin about 8, 10 or 12 days from the first time point.

In embodiments, the A2A receptor antagonist is administered at an amountof about 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200mg/kg or 300 mg/kg. In embodiments, the A2A receptor antagonist isadministered at an amount of about 1 mg/kg. In embodiments, the PD-1signaling pathway inhibitor is administered at an amount of less thanabout 1,300 mg. In embodiments, the PD-1 signaling pathway inhibitor isadministered at an amount of less than about 1,200 mg. In embodiments,the cancer is selected from lung cancer, bladder cancer, melanoma, renalcell carcinoma, colon cancer, ovarian cancer, gastric cancer, breastcancer, head and neck carcinoma, prostate cancer and a hematologicmalignancy.

Methods of Activating T Cells

In one aspect, a method of activating a T cell is provided. The methodincludes contacting the T cell with an A2A receptor antagonist, whereinthe A2A receptor antagonist is a compound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. n₁, n₂ andn₃ are independently an integer from 0 to 4. m₁, m₂ and m₃ areindependently an integer from 1 to 2. And v₁, v₂ and v₃ areindependently an integer from 1 to 2.

The A2A receptor antagonist provided herein is the same A2A receptorantagonist as described above for aspects of treating cancer using anA2A receptor antagonist and a PD-1 signaling pathway inhibitor.Therefore, the definitions for substituents and variables of formula (I)and (II) are the same as described above (e.g., R¹ is R^(1A)-substitutedfuranyl; R^(1A) is methyl; R² is —NR¹¹R¹²; R¹¹ and R¹² are independentlyhydrogen; R³ is R⁴-substituted C₁ alkyl; R⁴ is R⁵-substituted pyridinyl;R⁵ is R⁶-substituted 2 membered heteroalkyl; R⁶ is unsubstitutedtetrahydrofuranyl) and are incorporated herewith.

Thus, in embodiments, the A2A receptor antagonist is a compound offormula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the method includes contacting the T cell with a PD-1signaling pathway inhibitor. In embodiments, the PD-1 signaling pathwayinhibitor is an antibody or a small molecule. In embodiments, the T cellis an effector T cell or a natural killer cell. In embodiments, the Tcell is an adenosine-suppressed T cell. “An adenosine-suppressed T cell”is an effector T cell or a natural killer cell bound to adenosinethrough its A2A receptor, wherein the adenosine is bound in an amountsufficient to inhibit expression and/or secretion of immune responseactivating cytokines (e.g., expression of IL-2, IFN-γ or TNF). Inembodiments, the T cell is a CD8 T cell. In embodiments, the CD8 T cellis a CD45RA-negative CD8 T cell. In embodiments, the T cell is a CD4 Tcell. In embodiments, the CD4 T cell is a CD45RA-negative CD4 T cell. Inembodiments, the T cell is within a subject. In embodiments, the subjectis a cancer subject. In embodiments, the cancer subject is an anti-PD-1refractory subject.

Methods of Inhibiting A2A Receptor Activity

In one aspect, a method of inhibiting A2A receptor activity of a cell isprovided. The method includes contacting the cell with an A2A receptorantagonist, wherein the A2A receptor antagonist is a compound offormula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. n₁, n₂ andn₃ are independently an integer from 0 to 4. m₁, m₂ and m₃ areindependently an integer from 1 to 2. And v₁, v₂ and v₃ areindependently an integer from 1 to 2.

The A2A receptor antagonist provided herein is the same A2A receptorantagonist as described above for aspects of treating cancer using anA2A receptor antagonist and a PD-1 signaling pathway inhibitor.Therefore, the definitions for substituents and variables of formula (I)and (II) are the same as described above (e.g., R¹ is R^(1A)-substitutedfuranyl; R^(1A) is methyl; R² is —NR¹¹R¹²; R¹¹ and R¹² are independentlyhydrogen; R³ is R⁴-substituted C₁ alkyl; R⁴ is R⁵-substituted pyridinyl;R⁵ is R⁶-substituted 2 membered heteroalkyl; R⁶ is unsubstitutedtetrahydrofuranyl) and are incorporated herewith.

Thus, in embodiments, the A2A receptor antagonist is a compound offormula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the contacting includes binding the A2A receptorantagonist to an A2A receptor of the cell. In embodiments, the cell is aT cell. In embodiments, the T cell is an effector T cell or a naturalkiller cell. In embodiments, T cell is a CD8 T cell. In embodiments, theCD8 T cell is a CD45RA-negative CD8 Tcell. In embodiments, the T cell isa CD4 Tcell. In embodiments, the CD4 T cell is a CD45RA-negative CD4Tcell. In embodiments, the T cell is within a subject. In embodiments,the subject is a cancer subject. In embodiments, the cancer subject isan anti-PD-1 refractory subject.

Methods of Increasing Anti-Tumor Response

In one aspect, a method of increasing an anti-tumor immune response in asubject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist and a programmed cell death protein 1 (PD-1)signaling pathway inhibitor.

In another aspect, a method of increasing an anti-tumor immune responsein a subject in need thereof is provided. The method includesadministering to the subject a therapeutically effective amount of anadenosine-A2A (A2A) receptor antagonist, wherein the A2A receptorantagonist is a compound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. n₁, n₂ andn₃ are independently an integer from 0 to 4. m₁, m₂ and m₃ areindependently an integer from 1 to 2. And v₁, v₂ and v₃ areindependently an integer from 1 to 2.

The A2A receptor antagonist and the PD-1 signaling pathway inhibitorprovided herein are the same as described above for aspects of treatingcancer using an A2A receptor antagonist and a PD-1 signaling pathwayinhibitor. Therefore, the definitions for substituents and variables offormula (I) and (II) are the same as described above (e.g., R¹ isR^(1A)-substituted furanyl; R^(1A) is methyl; R² is —NR¹¹R¹²; R¹¹ andR¹² are independently hydrogen; R³ is R⁴-substituted C₁ alkyl; R⁴ isR⁵-substituted pyridinyl; R⁵ is R⁶-substituted 2 membered heteroalkyl;R⁶ is unsubstituted tetrahydrofuranyl) and are incorporated herewith.

In embodiments, the A2A receptor antagonist is a compound of formula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the method includes administering a therapeuticallyeffective amount of a PD-1 signaling pathway inhibitor. In embodiments,the PD-1 signaling pathway inhibitor is a PD-L1 antagonist. Inembodiments, the PD-L1 antagonist is a small molecule or an antibody.

Methods of Increasing CD8-Positive Cell Numbers

In one aspect, a method of increasing the amount of CD8-positive cellsrelative to the amount of regulatory T cells in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist and a programmed cell death protein 1 (PD-1) signalingpathway inhibitor.

In one aspect, a method of increasing the amount of CD8-positive cellsrelative to the amount of regulatory T cells in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein the A2A receptor antagonist is a compound offormula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. n₁, n₂ andn₃ are independently an integer from 0 to 4. m₁, m₂ and m₃ areindependently an integer from 1 to 2. And v₁, v₂ and v₃ areindependently an integer from 1 to 2.

The A2A receptor antagonist and the PD-1 signaling pathway inhibitorprovided herein are the same as described above for aspects of treatingcancer using an A2A receptor antagonist and a PD-1 signaling pathwayinhibitor. Therefore, the definitions for substituents and variables offormula (I) and (II) are the same as described above (e.g., R¹ isR^(1A)-substituted furanyl; R^(1A) is methyl; R² is —NR¹¹R¹²; R¹¹ andR¹² are independently hydrogen; R³ is R⁴-substituted C₁ alkyl; R⁴ isR⁵-substituted pyridinyl; R⁵ is R⁶-substituted 2 membered heteroalkyl;R⁶ is unsubstituted tetrahydrofuranyl) and are incorporated herewith.

In embodiments, the A2A receptor antagonist is a compound of formula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the method includes administering a therapeuticallyeffective amount of a PD-1 signaling pathway inhibitor. In embodiments,the PD-1 signaling pathway inhibitor is a PD-L1 antagonist. Inembodiments, the PD-L1 antagonist is a small molecule or an antibody.

Methods of Decreasing Tumor Volume

In one aspect, a method of decreasing tumor volume in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist and a programmed cell death protein 1 (PD-1) signalingpathway inhibitor.

In one aspect, a method of decreasing tumor volume in a subject in needthereof is provided. The method includes administering to the subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein the A2A receptor antagonist is a compound offormula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. n₁, n₂ andn₃ are independently an integer from 0 to 4. m₁, m₂ and m₃ areindependently an integer from 1 to 2. And v₁, v₂ and v₃ areindependently an integer from 1 to 2.

The A2A receptor antagonist and the PD-1 signaling pathway inhibitorprovided herein are the same as described above for aspects of treatingcancer using an A2A receptor antagonist and a PD-1 signaling pathwayinhibitor. Therefore, the definitions for substituents and variables offormula (I) and (II) are the same as described above (e.g., R¹ isR^(1A)-substituted furanyl; R^(1A) is methyl; R² is —NR¹¹R¹²; R¹¹ andR¹² are independently hydrogen; R³ is R⁴-substituted C₁ alkyl; R⁴ isR⁵-substituted pyridinyl; R⁵ is R⁶-substituted 2 membered heteroalkyl;R⁶ is unsubstituted tetrahydrofuranyl) and are incorporated herewith.

In embodiments, the A2A receptor antagonist is a compound of formula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the method includes administering a therapeuticallyeffective amount of a PD-1 signaling pathway inhibitor. In embodiments,the PD-1 signaling pathway inhibitor is a PD-L1 antagonist. Inembodiments, the PD-L1 antagonist is a small molecule or an antibody.

Methods of Enhancing Anti-Tumor Memory

In one aspect, a method of enhancing anti-tumor immune memory in asubject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist and a programmed cell death protein 1 (PD-1)signaling pathway inhibitor.

In one aspect, a method of enhancing anti-tumor immune memory in asubject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist, wherein the A2A receptor antagonist is acompound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. n₁, n₂ andn₃ are independently an integer from 0 to 4. m₁, m₂ and m₃ areindependently an integer from 1 to 2. And v₁, v₂ and v₃ areindependently an integer from 1 to 2.

The A2A receptor antagonist and the PD-1 signaling pathway inhibitorprovided herein are the same as described above for aspects of treatingcancer using an A2A receptor antagonist and a PD-1 signaling pathwayinhibitor. Therefore, the definitions for substituents and variables offormula (I) and (II) are the same as described above (e.g., R¹ isR^(1A)-substituted furanyl; R^(1A) is methyl; R² is —NR¹¹R¹²; R¹¹ andR¹² are independently hydrogen; R³ is R⁴-substituted C₁ alkyl; R⁴ isR⁵-substituted pyridinyl; R⁵ is R⁶-substituted 2 membered heteroalkyl;R⁶ is unsubstituted tetrahydrofuranyl) and are incorporated herewith.

In embodiments, the A2A receptor antagonist is a compound of formula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the method includes administering a therapeuticallyeffective amount of a PD-1 signaling pathway inhibitor. In embodiments,the PD-1 signaling pathway inhibitor is a PD-L1 antagonist. Inembodiments, the PD-L1 antagonist is a small molecule or an antibody.

In one aspect, a method of increasing global immune activation in asubject in need thereof is provided. The method includes administeringto said subject a therapeutically effective amount of an adenosine-A2A(A2A) receptor antagonist, wherein the A2A receptor antagonist is acompound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. n₁, n₂ andn₃ are independently an integer from 0 to 4. m₁, m₂ and m₃ areindependently an integer from 1 to 2. And v₁, v₂ and v₃ areindependently an integer from 1 to 2.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the method further includes administering atherapeutically effective amount of a PD-1 signaling pathway inhibitor.In embodiments, the PD-1 signaling pathway inhibitor is a PD-L1antagonist. In embodiments, the PD-L1 antagonist is a small molecule oran antibody. In embodiments, the method includes activating a CD4 T cellin the subject. In embodiments, the CD4 T cell is a memory T cell. Inembodiments, CD4 T cell is an effector T cell.

In embodiments, the relative amount of CD45RA-negative CD4 T cells inthe subject is increased. In embodiments, the relative amount of CD4 Tcells in the subject is increased. Where the relative amount of CD4 Tcells in the subject is increased the amount of CD4 T cells in thesubject can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more thanthat in a control. In certain instances, the increase is 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to acontrol. In embodiments, the relative amount of memory T cells in thesubject is increased. Where the relative amount of memory T cells in thesubject is increased the amount of memory T cells in the subject can be10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than that in acontrol. In certain instances, the increase is 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, or more in comparison to a control. Inembodiments, the relative amount of effector T cells in the subject isincreased. Where the relative amount of effector T cells in the subjectis increased the amount of effector T cells in the subject can be 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than that in a control.In certain instances, the increase is 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, or more in comparison to a control. In embodiments, themethod includes increasing the number of PD-1 positive cells in thesubject. Where the number of PD-1 positive cells in the subject isincreased the amount of PD-1 positive cells in the subject can be 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than that in a control.In certain instances, the increase is 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, or more in comparison to a control.

In embodiments, the method includes activating a CD8 T cell in thesubject. In embodiments, the relative amount of CD8 T cells in thesubject is increased. In embodiments, the relative frequency of TCRrecombination is increased. Where the relative frequency of TCRrecombination is increased the amounts of TCR recombination events canbe 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than that in acontrol. In certain instances, the increase is 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, or more in comparison to a control. Where thefrequency of TCR recombination is increased, the T cell receptorrepertoire (the number of T cells recognizing antigens that arechemically different from each other) is increased. Thus, the methodsprovided herein may increase the diversity of T cell clones in thesubject.

In embodiments, the subject is an anti-PD-1 refractory subject.

For the methods provided herein the A2A receptor antagonist may beadministered at an amount of about 100 mg BID. In embodiments, the A2Areceptor antagonist is administered for 28 consecutive days. Inembodiments, the A2A receptor antagonist is administered for 14consecutive days. In embodiments, the PD-1 signaling pathway inhibitoris administered at an amount of about 840 mg. In embodiments, the PD-1signaling pathway inhibitor is administered for 28 consecutive days. Inembodiments, the PD-1 signaling pathway inhibitor is administered for 14consecutive days. In further embodiments, the A2A receptor antagonistand the PD-1 signaling pathway inhibitor are administered on the sameday.

In embodiments, the A2A receptor antagonist is administered at a firsttime point and the PD-1 signaling pathway inhibitor is administered at asecond time point, wherein the first time point precedes the second timepoint. In embodiments, the second time point is within less than about120, 90, 60, 50, 40, 30, 28, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 days from said first time point. Inembodiments, the second time point is within less than about 120 days.In embodiments, the second time point is within less than about 90 days.In embodiments, the second time point is within less than about 60 days.In embodiments, the second time point is within less than about 50 days.In embodiments, the second time point is within less than about 40 days.In embodiments, the second time point is within less than about 30 days.In embodiments, the second time point is within less than about 28 days.In embodiments, the second time point is within less than about 20 days.In embodiments, the second time point is within less than about 19 days.In embodiments, the second time point is within less than about 18 days.In embodiments, the second time point is within less than about 17 days.In embodiments, the second time point is within less than about 16 days.In embodiments, the second time point is within less than about 15 days.In embodiments, the second time point is within less than about 14 days.In embodiments, the second time point is within less than about 13 days.In embodiments, the second time point is within less than about 12 days.In embodiments, the second time point is within less than about 11 days.In embodiments, the second time point is within less than about 10 days.In embodiments, the second time point is within less than about 9 days.In embodiments, the second time point is within less than about 8 days.In embodiments, the second time point is within less than about 7 days.In embodiments, the second time point is within less than about 6 days.In embodiments, the second time point is within less than about 5 days.In embodiments, the second time point is within less than about 4 days.In embodiments, the second time point is within less than about 3 days.In embodiments, the second time point is within less than about 2 days.In embodiments, the second time point is within less than about 2 days.In embodiments, the second time point is within less than about 1 day.

In embodiments, the second time point is within about 14 or 28 days fromthe first time point. In embodiments, the second time point is withinabout 14 days from the first time point. In embodiments, the second timepoint is within about 28 days from the first time point.

The methods provided herein including embodiments thereof may includeactivating a T cell in the subject. The methods provided hereinincluding embodiments thereof may include activating a CD4T cell in thesubject. In embodiments, the CD4 T cell is a memory T cell. Inembodiments, the CD4 T cell is an effector T cell. In embodiments, theCD4 T cell is a CD45RA-negative CD4 T cell. In embodiments, the relativeamount of a CD4 T cell is increased in the subject. In embodiments, therelative amount of an effector T cell is increased in the subject. Inembodiments, the relative amount of a CD45RA-negative CD4 T cell isincreased in the subject.

The methods provided herein including embodiments thereof may includeinhibiting A2A receptor activity of a cell in the subject. The methodsprovided herein including embodiments thereof may include increasing ananti-tumor immune response in a subject. The methods provided hereinincluding embodiments thereof may include increasing the amount ofCD8-positive cells relative to the amount of regulatory T cells in thesubject. The methods provided herein including embodiments thereof mayinclude enhancing anti-tumor immune memory in the subject. The methodsprovided herein including embodiments thereof may include enhancinganti-tumor immune memory in the subject. The methods provided hereinincluding embodiments thereof may include increasing global immuneactivation in the subject.

Pharmaceutical Compositions

Provided herein are pharmaceutical compositions including an A2Areceptor antagonist, a PD-1 signaling pathway inhibitor and apharmaceutically acceptable excipient. The provided compositions are,inter alia, suitable for formulation and administration in vitro or invivo. Suitable carriers and excipients and their formulations aredescribed in Remington: The Science and Practice of Pharmacy, 21stEdition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Bypharmaceutically acceptable carrier is meant a material that is notbiologically or otherwise undesirable, i.e., the material isadministered to a subject without causing undesirable biological effectsor interacting in a deleterious manner with the other components of thepharmaceutical composition in which it is contained. If administered toa subject, the carrier is optionally selected to minimize degradation ofthe active ingredient and to minimize adverse side effects in thesubject.

Compositions can be administered for therapeutic or prophylactictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease (e.g., cancer) in a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient.

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient (e.g. compositions describedherein, including embodiments or examples) is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. When administered in methods to treat a disease, the compoundsand antibodies described herein will contain an amount of activeingredient effective to achieve the desired result, e.g., modulating theactivity of a target molecule, and/or reducing, eliminating, or slowingthe progression of disease symptoms. Determination of a therapeuticallyeffective amount of a compound of the invention is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure herein.

Provided compositions can include a single agent or more than one agent.The compositions for administration will commonly include an agent asdescribed herein dissolved in a pharmaceutically acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofactive agent in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe subject's needs.

Solutions of the active compounds as free base or pharmacologicallyacceptable salt can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations can contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions can be delivered via intranasal or inhalablesolutions or sprays, aerosols or inhalants. Nasal solutions can beaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions can be prepared so that they aresimilar in many respects to nasal secretions. Thus, the aqueous nasalsolutions usually are isotonic and slightly buffered to maintain a pH of5.5 to 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations and appropriate drug stabilizers, ifrequired, may be included in the formulation. Various commercial nasalpreparations are known and can include, for example, antibiotics andantihistamines.

Oral formulations can include excipients as, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. In someembodiments, oral pharmaceutical compositions will comprise an inertdiluent or assimilable edible carrier, or they may be enclosed in hardor soft shell gelatin capsule, or they may be compressed into tablets,or they may be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compounds may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 75% of the weight of the unit, or preferably between25-60%. The amount of active compounds in such compositions is such thata suitable dosage can be obtained.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered and the liquid diluent firstrendered isotonic with sufficient saline or glucose. Aqueous solutions,in particular, sterile aqueous media, are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. For example, one dosage could be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion.

Sterile injectable solutions can be prepared by incorporating the activecompounds or constructs in the required amount in the appropriatesolvent followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium.Vacuum-drying and freeze-drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredients, can be usedto prepare sterile powders for reconstitution of sterile injectablesolutions. The preparation of more, or highly, concentrated solutionsfor direct injection is also contemplated. DMSO can be used as solventfor extremely rapid penetration, delivering high concentrations of theactive agents to a small area.

The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials. Thus, thecomposition can be in unit dosage form. In such form the preparation issubdivided into unit doses containing appropriate quantities of theactive component. Thus, the compositions can be administered in avariety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms suitable for oraladministration include, but are not limited to, powder, tablets, pills,capsules and lozenges.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated (e.g. symptoms of cancer and severity of such symptoms),kind of concurrent treatment, complications from the disease beingtreated or other health-related problems. Other therapeutic regimens oragents can be used in conjunction with the methods and compounds of theinvention. Adjustment and manipulation of established dosages (e.g.,frequency and duration) are well within the ability of those skilled inthe art.

For any composition (e.g., the compounds and antibodies provided)described herein, the therapeutically effective amount can be initiallydetermined from cell culture assays. Target concentrations will be thoseconcentrations of active compound(s) that are capable of achieving themethods described herein, as measured using the methods described hereinor known in the art. As is well known in the art, effective amounts foruse in humans can also be determined from animal models. For example, adose for humans can be formulated to achieve a concentration that hasbeen found to be effective in animals. The dosage in humans can beadjusted by monitoring effectiveness and adjusting the dosage upwards ordownwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention should be sufficient to affect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the artand include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate and the like.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

In one aspect, a pharmaceutical composition including an A2A receptorantagonist, a PD-1 signaling pathway inhibitor and a pharmaceuticallyacceptable excipient is provided.

In embodiments, the A2A receptor antagonist is a compound of formula:

In formula (I), R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN,—SO₂Cl, —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,—NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹, —C(O)—OR⁹,—C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² isindependently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl, —SO_(n2)R¹¹,—SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂, —NHC═(O)NR¹¹R¹²,—N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹, —C(O)—OR¹¹, —C(O)NR¹¹R¹²,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R³ is independently hydrogen,halogen, —CX^(c) ₃, —CN, —SO₂Cl, —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂,—ONR¹³R¹⁴, —NHC═(O)NHNH₂, —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴,—NH—O—R¹³, —C(O)R¹³, —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ areindependently hydrogen, halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I. n₁, n₂ andn₃ are independently an integer from 0 to 4. m₁, m₂ and m₃ areindependently an integer from 1 to 2. And v₁, v₂ and v₃ areindependently an integer from 1 to 2.

The A2A receptor antagonist and the PD-1 signaling pathway inhibitorprovided herein are the same as described above for aspects of treatingcancer using an A2A receptor antagonist and a PD-1 signaling pathwayinhibitor. Therefore, the definitions for substituents and variables offormula (I) and (II) are the same as described above (e.g., R¹ isR^(1A)-substituted furanyl; R^(1A) is methyl; R² is —NR¹¹R¹²; R¹¹ andR¹² are independently hydrogen; R³ is R⁴-substituted C₁ alkyl; R⁴ isR⁵-substituted pyridinyl; R⁵ is R⁶-substituted 2 membered heteroalkyl;R⁶ is unsubstituted tetrahydrofuranyl) and are incorporated herewith.

In embodiments, the A2A receptor antagonist is a compound of formula:

In formula (II), R⁶, R^(6.1) and R^(6.2) are independently hydrogen,halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the A2A receptor antagonist is a compound of formula:

In embodiments, the PD-1 signaling pathway inhibitor is a programmeddeath-ligand 1 (PD-L1) antagonist or a PD-1 antagonist. In embodiments,the programmed death-ligand 1 (PD-L1) antagonist is an antibody or asmall molecule. In embodiments, the PD-L1 antagonist is an antibody. Inembodiments, the antibody is atezolizumab. In embodiments, the PD-1antagonist is an antibody or a small molecule. In embodiments, the A2Areceptor antagonist and the PD-1 signaling pathway inhibitor are presentin a combined synergistic amount, wherein the combined synergisticamount is effective to treat cancer in a subject in need thereof.

In embodiments, the pharmaceutical composition is in oral dosage form.In embodiments, the adenosine-A2A (A2A) receptor antagonist (e.g,CPI-444) is presented as size 0-elongated hydroxypropyl methylcellulose(HPMC) capsules containing adenosine-A2A (A2A) receptor antagonistcompound (e.g, CPI-444) at 10 mg, 25 mg or 100 mg, as a dry powdermixture of adenosine-A2A (A2A) receptor antagonist compound (e.g,CPI-444) resinate with common excipients and packaged in high densitypolyethylene (HDPE) bottles fitted with a polypropylene tamper evidentchild-resistant cap with an integrated desiccant. The adenosine-A2A(A2A) receptor antagonist (e.g, CPI-444) resinate is a complex of theadenosine-A2A (A2A) receptor antagonist and a cation exchange resin(Amberlite IRP69™). The ingredients are listed in Table A.

TABLE A CPI-444 Capsules Table of Ingredients Ingredient FunctionCPI-444 (adenosine-A2A (A2A) Active ingredient receptor antagonist)Sodium polystyrene sulfonate Ion-exchange resin, release modifying(Amberlite IRP69) resin agent Mannitol, spray-dried DiluentCroscarmellose sodium Disintegrant Colloidal silicon dioxide GlidantSodium stearylfumarate Lubricant Size 0-elongated HPMC capsule Capsuleshell:  10 mg - opaque, Swedish orange  25 mg - opaque white 100 mg -opaque, Swedish orangeDetecting Adenosine Receptor Activation

Cyclic AMP (cAMP) response element binding protein (CREB) is a cellulartranscription factor. CREB is activated by signaling cascades resultantfrom an array of extracellular signals. One such activating signalcascade is triggered by agonist binding to adenosine receptor (e.g. A2Aand A2B receptors). Agonist activation of adenosine receptor results inactivation of CREB by phosphorylation. Agonist activation of adenosinereceptor also results in activation of protein kinase A (PKA) upstreamof CREB.

Cancer therapies described above which include adenosine receptorantagonists, alone or in combination, alter signaling cascades thatresult in CREB activation (see FIG. 24). Detection of downstream effectsof treatment with adenosine receptor antagonists can be assayed todetermine cellular response to treatment. Activation of CREB activationcan be detected by detection of phosphorylated CREB.

In embodiments, pCREB is detected by a pCREB detection agent. Inembodiments, pCREB is detected by an antibody (e.g. a commerciallyavailable antibody). In embodiments, a pCREB detection agent is detectedby Fluorescence-activated cell sorting (FACS). In embodiments, pCREB isdetected in a subpopulation of cells (e.g. T cells and/or B cells). Inembodiments, pCREB is detected using an antibody in an ELISA format. Inembodiments, ELISA detection can be from bulk cell lysate or sorted Bcells and/or T cells.

In embodiments, cells for detection of pCREB can be harvested from blood(e.g. from circulating blood). In embodiments, cells for detection ofpCREB can be harvested from a tumor site. In embodiments, cells for thedetection of pCREB are isolated, stained and fixed. In embodiments, cellstaining is with antibodies against pCREB, CD3, CD4, CD8, CD27, CD20,CD45RA, cPARP. In embodiments, cells for the detection of pCREB aresorted by FACs. In embodiments, FACS detection of an antibody againstCD19 and an antibody against CD20 indicate a B cell. In embodiment, FACSdetection of antibodies against CD3, CD4 and CD8 indicates a T cell. Inembodiments, FACS detection of antibodies against cPARP indicates anapoptotic cell. In embodiments, detection of pCREB induction is from anisolated cell population (e.g. B cells or T cells).

In embodiments, detection of activated PKA is used in addition to, or asa proxy for, detection of pCREB.

CREB activation can be induced by activation of adenosine receptoragonists including adenosine, NECA, or analogs thereof. NECA is asynthetic adenosine analog. In embodiments, NECA is administered tocells in a concentration of about 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5μM, 1.0 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 10 μM, 15 μM, 20 μM, 30 μM ormore to activate adenosine receptors.

In embodiments, inhibition of pCREB induction by an adenosine receptoragonist can be used as an in vitro screening or evaluation assay toidentify and characterize adenosine receptor antagonists.

Patient Selection and Dosage Adjustment

In patients treated with adenosine receptor antagonists, alone or incombination, effects on downstream effectors (e.g. CREB) can be used todetermine treatment or dosage efficacy of the adenosine receptorantagonist therapy. Furthermore, assessment of CREB activation can beused to determine the diurnal timing of therapy administration.

An individual patient reaction to treatment with an adenosine receptorantagonist (e.g. an A2A receptor or A2B receptor antagonist) can bedetected by measuring of cellular effects. In embodiments, cellulareffects of treatment can be monitored in a patient sample (e.g. a bloodor tumor sample). In embodiments, a blood sample is used to assay CREBactivation. As described above adenosine receptor agonists result inCREB activation, conversely adenosine receptor antagonists can inhibitthe activation of CREB. In embodiments, monitoring inhibition ofactivation of CREB via the adenosine receptor pathway can indicateefficacy of an adenosine receptor antagonist.

In embodiments, cells are isolated from a patient sample (e.g. a bloodor tumor sample). In embodiments, CREB activation following treatmentwith an adenosine receptor agonist (e.g. NECA) is monitored prior totreatment with an adenosine receptor antagonist (e.g. detection ofinduction of pCREB prior to treatment with CPI-444). In embodiments,pCREB induction by an adenosine receptor agonist is assayed relative toa control sample (e.g. cells treated with PMA). In embodiments, anadditional sample is collected following treatment with an adenosinereceptor antagonist, alone or in combination (e.g. CPI-444, or CPI-444combination therapy with azetolizumab). In embodiments, a sample iscollected after about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks or more followingtreatment with an adenosine receptor antagonist. A comparison can bemade between pCREB induction by an adenosine receptor agonist prior toand following treatment with an adenosine receptor antagonist todetermine the degree to which treatment has reduced downstream effectsof adenosine receptor activation (e.g. pCREB induction).

In embodiments, subjects who display attenuated induction of pCREB by anadenosine receptor agonist following treatment with an adenosinereceptor antagonist are selected as responsive to treatment withadenosine receptor antagonists. In embodiments, where attenuation orinhibition of pCREB induction by NECA by an adenosine receptorantagonist is incomplete a dosage of the adenosine receptor antagonistcan be increased. In embodiments, a patient sample is taken prior toadenosine pathway blockade (e.g. with an adenosine receptor antagonist)and treated with an adenosine receptor agonist to determine the level ofinduced pCREB signaling which may guide patient selection for treatmentby adenosine pathway blockade (e.g. with an adenosine receptorantagonist).

Furthermore, diurnal variations in concentration of an adenosinereceptor antagonist in the circulation result from one or twice dailyadministration. This variation in concentration throughout the course ofthe day can impact treatment efficacy. Using methods and compositions ofthe present invention variations of the efficacy of treatment can bemonitored by detecting pCREB induction by an adenosine receptor agonistat different time points following administration of an adenosinereceptor antagonist. In embodiments, pCREB induction by an adenosinereceptor agonist can be monitored at about 0 hours, 0.5 hours, 1 hours,2 hours 3 hours, 4 hours, 5 hours, 8 hours, 9 hours, 10 hours, 12 hours,14 hours, 16 hours, 20 hours, or 24 hours following administration of anadenosine receptor antagonist. In embodiments, timing of administrationof an adenosine receptor antagonist can be altered for maximalinhibition of pCREB induction by an adenosine receptor agonist.

EXAMPLES Example 1

Binding Affinity for Human Adenosine Receptors (V81444-07-076)

The ability of CPI-444 to displace radioligand binding for the fouridentified adenosine receptor subtypes (A1, A2A, A2B, and A3) was testedin human recombinant receptors expressed in mammalian cell lines.Results are shown in Table 1.

TABLE 1 Displacement of radioligand binding by CPI-444 Receptor pKi MeanKi nM Selectivity against A_(2A) Adenosine A₁ 6.72 192 ×54 AdenosineA_(2A) 8.45 3.54 Adenosine A_(2B) 5.82 1,528 ×431 Adenosine A₃ 5.612,455 ×693CPI-444 bound A2A receptors with an affinity (Ki) value of 3.54 nM (thenegative logarithm of Ki [pKi]=8.45). CPI-444 showed greater than 50fold selectivity for the A2A receptor over other adenosine receptorsubtypes.

Functional Activity on Human Adenosine Receptors (V81444-07-078)

CPI-444 was evaluated in experimental paradigms designed to quantifyantagonist interactions with the four identified human adenosinereceptor subtypes expressed in Chinese hamster ovary (CHO-K1) cells. Atall concentrations tested, CPI-444 caused a right-shift in the agonistconcentration-response curve without decreasing the maximum agonistresponse, indicating a competitive mode of action. Antagonist pA2(negative logarithm of the antagonist concentration causing a 2-foldshift in the agonist concentration response curve [equivalent to 50%occupancy]) values were estimated from the extent of this rightshift andshowed V81444 to be a potent A2A receptor antagonist with a pA2 value of8.49 (3.2 nM) at the A2A receptor (Table 2).

TABLE 2 CPI-444 adenosine receptor antagonist activity. pA₂ pA₂expressed as chemical Selectivity against Receptor mean concentration nMA_(2A) Adenosine A₁ 6.53 295 ×92 Adenosine A_(2A) 8.49 3.2 AdenosineA_(2B) 6.36 436 ×136 Adenosine A₃ 5.65 2,240 ×700CPI-444 was more than 90-fold selective for the A2A receptor relative tothe other adenosine receptors.

Effect of CPI-444 on cAMP Production (CPI-RSR-003)

Adenosine signaling through A2AR leads to increases in the levels ofcAMP. This study evaluated the ability of CPI-444 to prevent cAMPproduction in primary human T cells stimulated with NECA, a stableanalog of adenosine (CPI-RSR-003).

T cells were isolated from human PBMC by negative selection andactivated via CD3/CD28 stimulation for 48 hours to induce A2ARexpression. Stimulated T cells were then “rested” for 24 hours byremoval of CD3/CD28 stimulation in order to minimize background levelsof cAMP. Rested T cells were incubated in the presence of NECA andCPI-444 or vehicle control for 10 minutes prior to measurement of cAMPusing the LANCE Ultra cAMP FRET-based assay (Perkin Elmer). CPI-444completely blocked the production of cAMP upon NECA treatment at alllevels of NECA tested (10-5 to 10-9 M). CPI-444 also prevented cAMPproduction upon NECA stimulation in a dose-dependent manner (FIG. 6).These results confirm that CPI-444 is an A2AR antagonist capable ofinhibiting cAMP induced by adenosine signaling.

Effect of CPI-444 on IL-2 and IFNγ Secretion (CPI-RSR-002)

The objective of this study was to determine if CPI-444 abrogates theimmunosuppressive effects of adenosine on T cell activation and Th1cytokine release in vitro (CPI-RSR-002). Primary human PBMCs werecultured for 1 hour in the presence of an A2AR agonist (NECA orCGS21680, 1 μM) to simulate the effects of adenosine on immune cellfunction. Purified anti-CD3 and anti-CD28 monoclonal antibodies (1ug/ml) were then added to activate T cells for 48 hours. In this study,AlphaLISA assays (PerkinElmer) analyzed on an EnVision MultiLabel Readerwere used to measure cytokine release according to the manufacturer'sinstructions. NECA and CGS21680 suppressed release of the Th1 cytokinesIL-2 and IFNγ, mimicking the immunosuppressive effects of adenosinesignaling (FIG. 7).

Blockade of A2AR with CPI-444 (1 μM) prior to T cell activationneutralized the immunosuppressive effects of NECA and CGS21680 andrestored IL-2 and IFNγ secretion back to levels observed in the absenceof exogenous adenosine signaling (DMSO control). These results show thatrestoration of T cell function is an important mechanism by whichCPI-444 enables an anti-tumor response in vivo.

CPI-444 does not Inhibit Tumor Cell Proliferation In Vitro(CPI444-RSR-006)

CPI-444 inhibits the growth of MC38, CT26, and EL4 tumors at eitherprimary (MC38, CT26) or metastatic (EL4) sites in syngeneic mouse tumormodels. This study evaluated the effects of CPI-444 on mouse tumor cellproliferation and viability. MC38, CT26, and EL4 cells were cultured inthe presence of CPI-444 at a concentrations ranging from 10 □M to 1 pMfor 24 hours. Staurosporine, a well-characterized inducer of apoptosis,was included as a positive control for cell death. Cellviability/proliferation was measured by XTT. In this assay, XTT saltsare cleaved by metabolically active (viable) cells, thereby producing acolorimetric change in the culture media that can be quantified bymeasuring absorbance at 405 nm and 620 nm on a spectrophotometer. Nosignificant decrease in the Specific Absorbance(A450Test—A450Blank—A620Test) was observed in MC38, CT26, or EL4cultures at any concentration of CPI-444 tested (representative results,FIG. 8). These results indicate that CPI-444 efficacy observed in vivois likely not due to a direct effect on tumor cell proliferation.

Effect of CPI-444 on pERK Levels in Human CD4+ Cells (CPI-RSR-008)

This study shows that stimulation of A2AR with an adenosine analog(NECA) dampens ERK activation in human PBMCs following TCR cross-linking(CPI-RSR-008). Both CPI-444 and the A2AR specific antagonist ZM 241385restore ERK signaling in the presence of NECA. The percentage of CD4+ Tcells showing TCR-mediated ERK phosphorylation was reduced in thepresence of NECA (1 μM). Addition of CPI-444 restored pERK levels in adose-dependent manner (FIG. 9). This finding supports a role for CPI-444in restoring T cell activation in the presence of otherwiseimmunosuppressive levels of adenosine.

Effect of CPI-444 on Phosphorylation of cAMP Response Element BindingProtein (pCREB; CPI-RSR-007)

Adenosine signaling via A2AR leads to an increase in intracellular cAMPand subsequent phosphorylation of CREB. This study demonstrates that theadenosine analog NECA activates phosho-CREB in fresh PBMCs, primarily inthe B cell population (CPI-RSR-007). Furthermore, this phosphorylationevent is completely inhibited by CPI-444, as well as by the known A2ARantagonist ZM 241385 (FIG. 10). This finding demonstrates that CPI-444inhibits NECA-mediated cell signaling through A2AR and provides afunctional assay for CPI-444 activity.

In Vivo Studies

Oral administration of CPI-444 at 100 mg/kg or 10 mg/kg significantlyinhibits the growth of MC38 colon tumors compared to the vehicle controlin syngeneic hosts.

Oral administration of CPI-444 at 10, 30 or 100 mg/kg produced atherapeutic response on established primary tumors in the EL4 syngeneicmouse lymphoma model. A significant dose-dependent inhibition of tumorgrowth within regional lymph nodes was observed in mice treated withCPI-444.

CPI-444 (100 mg/kg) or anti-PD-1 antibody monotherapy inhibits thegrowth of CT26 colon tumors in syngeneic hosts. CPI-444+anti-PD-1combination therapy eliminated CT26 tumors in nearly all mice.Combination therapy also produced a significant increase in long-termsurvival compared to either agent administered alone.

Syngeneic EL4 Mouse Lymphoma Model (CPI-RSR-001)

This study evaluated the anti-tumor effect of CPI-444 on tumor growthand metastasis in a transplanted CD4+ mouse T cell lymphoma model(CPI-RSR-001). Syngeneic C57BL/6 female mice (8-10 weeks old) wereinjected (via subcutaneous route) with EL4 cells. Tumor-bearing micewere administered control vehicle (40% Hydroxypropyl Beta-Cyclodextrin)or CPI-444 solution daily by oral gavage upon formation of measurabletumors (140±55 mm3). CPI-444 doses of 10, 30, and 100 mg/kg wereevaluated. CPI-444 treatment produced a minimal therapeutic response onestablished primary tumors. A dose response was observed, yet all doselevels failed to produce a significant inhibition of tumor growth. Incontrast, a significant, dose-dependent decrease in the number and sizeof enlarged regional lymph nodes was observed (FIG. 11), indicating thatCPI-444 inhibited or eliminated tumor metastases in this model.

Syngeneic MC38 Mouse Colon Carcinoma Model (CPI-RSR-004)

The objective of this study was to evaluate the anti-tumor activity ofCPI-444 in a mouse colon carcinoma model (CPI-RSR-004). MC38 coloncancer cells were subcutaneously injected onto the backs of syngeneicC57BL/6 mice. One day after tumor cell engraftment, vehicle control (40%Hydroxypropyl Beta-Cyclodextrin) or CPI-444 was administered daily viaoral gavage for 28 days. Administration of CPI-444 at 1 mg/kg did notinhibit tumor growth, however doses of 10 mg/kg and 100 mg/kg resultedin a significant inhibition of tumor growth (FIG. 12). Notably, completetumor regression was observed in a subset of mice within all cohortstreated with CPI-444 (FIG. 12). It is possible that full tumoreradication could be achieved in additional mice with longeradministration of CPI-444. These results demonstrate that MC38 isresponsive to CPI-444 treatment. Syngeneic CT26 Mouse Colon Cancer Modelwith CPI-444 in Combination with anti-PD1 (CPI-RSR-005)

The objective of this study was to evaluate the effects of CPI-444 in atransplanted mouse colon cancer model in combination with a blockinganti-PD-1 monoclonal antibody (CPI-RSR-005). CT26 mouse colon cancercells were engrafted onto the back of syngeneic male Balb/c mice. Oraladministration of control vehicle (40% solution ofhydroxypropyl-beta-cyclodextrin) or CPI-444 (100 mg/kg) was initiatedthe same day tumors were engrafted (Day 0). Treatment continued for 12days. Half of the mice in the vehicle control group as well as half themice in the CPI-444 treatment group received anti-PD-1 mAb (RMP1-14, 100ug/mouse, i.p.) on days 7, 9, 11, and 13. Administration of anti-PD-1 orCPI-444 resulted in an inhibition of tumor growth, however tumors werenot completely eradicated by either treatment (FIG. 13). Administrationof CPI-444 in combination with anti-PD-1 stabilized or eliminated tumorsin 8/9 mice, resulting in improved overall survival for more than 3weeks following the last dose of CPI-444 or anti-PD-1 antibody (FIG.13).

Example 2

Biomarker and Clinical Activity of CPI-444, a Novel Small MoleculeInhibitor of A2A Receptor (A2AR), in a Ph1b Study in Advanced Cancers

Adenosine is immunosuppressive and is produced at high concentrations intumors by both CD73 and direct release from tumor cells. Adenosineactivates A2AR, an immune checkpoint that leads to direct suppression ofeffector T cells and stimulation of regulatory T cells. CPI-444 is anoral, selective A2AR inhibitor that has been well tolerated in Phase(Ph) 1 and 2 studies in non-oncology indications. CPI-444 shows activityin multiple preclinical tumor models as a single agent and synergisticefficacy when given in combination with other checkpoint inhibitors,including anti-PD-L1.

CPI-444, with or without the investigational agent atezolizumab(anti-PD-L1), is being studied in an ongoing Ph1b trial in solid tumorpatients (pts). Pts with either lung, melanoma, triple negative breast,bladder, colorectal, renal, or head and neck cancers are treated atvarious doses of either single agent CPI-444 or combined withatezolizumab. After a dose selection stage, pts are treated in 10disease specific cohorts (5 single agent and 5 combination). Cohorts maybe expanded based on response criteria: complete response, partialresponse or stable disease (SD). Biomarkers are evaluated includingimmune cells by flow cytometry in peripheral blood and pre/posttreatment tumor biopsies as well as adenosine pathway modulation byimmunohistochemistry and gene expression.

In 7 pts treated to date, CPI-444 has been well tolerated with no Grade3 or 4 treatment related adverse events. 2 pts (1 combination and 1single agent) have reached the first efficacy assessment by CT and bothdemonstrated SD (unconfirmed at 2 months); these 2 pts, and 4 others whohave not yet reached efficacy evaluation, remain on treatment.

In the two pts with SD, peripheral blood showed increases in PD-1+CD8+cells (1.7 and 2.4 fold compared to baseline). This is consistent withpreclinical models and reflects effector T cell activation, similar toreports by others in patients treated with anti-PD-L1.

CPI-444 is well tolerated and demonstrates biological activityindicating activation of T cell immunity. This is the firstdemonstration of treatment-associated immune modulation in cancerpatients receiving an adenosine antagonist.

Example 3

Adenosine A2A Receptor Antagonist, CPI-444, Blocks Adenosine-Aediated TCell Suppression and Exhibits Anti-Tumor Activity Alone and inCombination with Anti-PD-1 and Anti-PD-L1

Elevated extracellular adenosine in the tumor microenvironment generatesan immunosuppressive niche that promotes tumor growth and metastasis.Adenosine signaling via A2A receptor (A2AR) on immune cells suppressesanti-tumor immunity and may also limit efficacy of immunotherapies suchas anti-PD-L1 and anti-PD-1 antibodies.

CPI-444 is a potent, oral, selective A2AR antagonist that has been welltolerated in Ph 1 and 2 studies in non-oncology indications. Efficacy ofCPI-444 was evaluated in MC38 and CT26 syngeneic mouse tumor models. InMC38, daily treatment of mice with CPI-444 (1, 10, 100 mg/kg) led todose-dependent inhibition of tumor growth, leading to tumor eliminationin 9/30 mice. Combining CPI-444 with anti-PD-L1 treatment in MC38synergistically inhibited tumor growth and eliminated tumors in 90% oftreated mice. In an additional model, CT26, CPI-444 alone or anti-PD-1alone led to non-significant reductions in tumor growth; however, thecombination of CPI-444 and anti-PD-1 led to a synergistic inhibition oftumor growth and prolonged survival compared to either agent alone.

When cured mice were later re-challenged with MC38 cells, tumor growthwas fully inhibited, indicating that CPI-444 induced systemic anti-tumorimmune memory. CD8⁺ T cell depletion abrogated the efficacy ofCPI-444±anti-PD-L1 treatment, demonstrating a role for CD8⁺ T cells inmediating primary and secondary immune responses.

Anti-tumor efficacy of CPI-444±anti-PD-L1 was associated with increasedCD8⁺ cell infiltration and activation in MC38 tumor tissues.Additionally, levels of immune checkpoints were modulated by treatmentwith CPI-444, including GITR, OX40, and LAG3 on tumor infiltratinglymphocytes and circulating T cells, suggesting a broad role foradenosine mediated immunosuppression.

Based on these results and others, Applicants have initiated a Phase 1bclinical trial to examine safety, tolerability, biomarkers, andpreliminary efficacy of CPI-444 as a single agent and in combination theinvestigational anti-PD-L1 antibody, Atezolizumab, in patients withsolid tumors.

Example 4

CPI-444: A Potent and Selective Inhibitor of Adenosine 2A Receptor(A2AR) Induces Anti-Tumor Responses Alone and in Combination withAnti-PD-L1.

Adenosine is immune-suppressive, acting through adenosine 2A receptor(A2AR) which is expressed on cytotoxic, helper and regulatory T cells,as well as NK, dendritic and myeloid derive suppressor cells. CPI-444 isan oral, selective inhibitor of A2AR that is active as a single agent inmultiple syngeneic mouse models and is synergistic when combined withanti-PD-1 or anti-PD-L1 antibodies in these models. 75 subjects werepreviously dosed with CPI-444 in non-oncology trials, and CPI-444 wellwas well tolerated with no significant adverse events noted. A Phase1/1b study was initiated that explores safety and efficacy of CPI-444 asa single agent as well as in combination with the anti-PD-L1 antibodyTECENTRIQ® (atezolizumab) in selected histologies.

In step 1 of the trial, patients were dosed with either 100 mg BID for14 days out of a 28 day cycle, 100 mg BID for 28 days, 200 mg QD for 14days or 50 mg or 100 mg BID for 14 days in combination with TECENTRIQ®(840 mg Q2W). Pharmacodynamic analysis was conducted on peripheral bloodcells to inform dose selection. Step 1 of Phase1/1b was fully enrolled(n=48) and dose was selected (100 mg BID) based on pharmacodynamicanalysis of A2AR pathway. Complete inhibition observed at 100 mg BIDdose that is sustained in 28 day continuous dosing cohort. Increases inactivated CD8 cell frequencies were observed in patients treated withsingle agent CPI-444 and combined with TECENTRIQ®, suggesting immuneactivation in response to treatment (FIG. 19A). TCR repertoire changeswere induced in peripheral blood by single agent CPI-444 in patientsubsets, including patients refractory to prior anti-PD-1 therapy (FIGS.20A-20C).

High TCR diversity (low clonality) at baseline and changes in TCRrepertoire following treatment show association with early efficacy datain anti-PD-1 naïve and refractory patients. A similar rate of stabledisease was observed in anti-PD-1 refractory patients and in both PD-L1positive and negative patient subsets (FIGS. 21A and 21B). This is thefirst demonstration of immune cell activation and anti-tumor activity inpatients receiving an adenosine antagonist.

Example 5

MC38 mouse colon cancer cells were engrafted onto the back of syngeneicC57BL/6 mice. Oral administration of control vehicle or CPI-444 (100mg/kg) was initiated 9 days after tumors were engrafted (Day 0).Treatment continued for 12 days. Half of the mice in the vehicle controlgroup as well as half the mice in the CPI-444 treatment group receivedanti-PD-L1 mAb (10F.9G2, 200 ug/mouse, i.p.) on days 9, 12, 15, and 18.100 ug of Anti-mCD4 (Clone GK1.5) was administered on days 8, 11, 14,and 17, and 500 ug of Anti-mCD8 (Clone 53-6.72) was administered on days8 and 15. T cell depletion was verified by flow analysis. FIGS. 23A and23B show tumor volume at different time points since engraftment for thedosing cohorts. These results suggest CD8+ T cells are required for theefficacy of CPI-444 alone or in combination with Anti-PD-L1.

Example 6

MC38 mouse colon cancer cells were engrafted onto the back of syngeneicC57BL/6 mice. Oral administration of control vehicle or CPI-444 (100mg/kg) was initiated 7 days after tumors were engrafted (Day 0) (FIG.23C). Treatment continued for more than 9 days. Half of the mice in thevehicle control group as well as half the mice in the CPI-444 treatmentgroup received anti-PD-L1 mAb (10F.9G2, 200 ug/mouse, i.p.) on days 7,10, 13, and 16. 100 ug of Anti-mCD4 (Clone GK1.5) and/or 500 ug ofAnti-mCD8 (Clone 53-6.72) was administered on day 6. T cell depletionwas verified by flow analysis. FIGS. 23A and 23B show tumor volume atdifferent time points since engraftment for the dosing cohorts. Theseresults suggest CD8+ T cells are required for the efficacy of CPI-444alone or in combination with Anti-PD-L1.

Example 7

Materials and Methods

Whole Blood Processing

Blood samples were derived from patients and processed using thefollowing protocol:

-   -   Whole blood in heparin delivered overnight and assay begins the        next morning    -   Aliquot 67.5 uL blood per well and recover at 37 C for 1 hr    -   Add 7.5 uL of NECA or PMA per well for 15 minutes at 37 C        -   NECA at 1, 3 or 10 uM    -   Fix cells with 1.5 mL of BD Lyse/Fix buffer according to        manufacturer    -   Spin and resuspend in 1 mL cold MeOH and store −80 C.        Antibody Staining

Fix cells derived from patient blood were stained using the followingprotocol:

-   -   Spin out of MeOH    -   Wash 2× with FACS buffer (phosphate buffered saline containing        1% bovine serum albumin and 0.1% sodium azide)    -   Stain 1 hour        -   Antibody cocktail:            -   pCREB Alexa Flour647 (Cell Signaling Technology Cat. No.                14001S)            -   CD3 Horizon V500 (BD Cat. No. 561416)            -   CD4 Brilliant Violet 421 (BD Cat. No. 562424)            -   CD8 PerCP-Cy5.5 (BD Cat. No. 560662)            -   CD27 FITC (BD Cat. No. 340424)            -   CD20 PE (BD Cat. No. 561174)            -   CD45RA PE-Cy7 (BD Cat. No. 649457)            -   cPARP Alexa Flour700 (BD Cat. No. 560640)    -   Wash 2× with FACS buffer    -   Fix cells with 1.6% paraformaldehyde (PFA) for 5 minutes at        ambient temperature    -   Spin, aspirate, and bring cells to acquisition volume in 1.6%        PFA    -   Acquire cells on flow cytometer.

Example 8

Induction of pCREB by NECA in B cells harvested from whole blood andstained as described above was monitored. Two subjects were assayed, onetreated with 200QD CPI-444 (FIG. 27) and a subject treated with 50BIDCPI-444+Atezolizumab (atezo) (FIG. 28) prior to treatment and at 2 timepoints after 14 days of treatment. NECA was used to stimulate CREBactivation via the adenosine receptor pathway in concentrations of 1 uMNECA, (sub-saturation, where inhibition is expected), and 10 uM NECA(saturation, where NECA may out-compete CPI-444 resulting ininhibition). Control samples were treated with phorbol myristate acetate(PMA).

Baseline induction of pCREB following NECA treatment is shown at C1D1prior to treatment. NECA induction of pCREB was then monitored at C1D14at 0 hr (trough) prior to CPI-444 or CPI-444+atezo treatment, whenCPI-444 concentration in circulating blood is at its lowest (see FIG.26). The second time point is C1D14, 1.5 hours after administration ofCPI-444 or CPI-444+atezo treatment.

In FIG. 27, partial inhibition of the adenosine receptor activation byCPI-444 is seen by the inhibition of the NECA induced increase of pCREBin B cells treated with 1 μM NECA. In FIG. 28, near complete inhibitionof adenosine receptor activation by CPI-444+atezo is seen by theinhibition of the NECA induced increase of pCREB in B cells treated with1 μM NECA.

Example 9

Induction of pCREB by NECA in B cells (FIG. 29 and FIG. 30) and T cells(FIG. 31) harvested from whole blood and stained as described above wasmonitored. Two subjects were assayed, one treated with 200QD CPI-444 anda subject treated with 50BID CPI-444+Atezolizumab (atezo) prior totreatment and at multiple time points after 14 days of treatment. NECAwas used to stimulate CREB activation via the adenosine receptor pathwayin concentrations of 1 uM, 3 uM, and 10 uM. Unstimulated control sampleswere treated with phorbol myristate acetate (PMA).

pCREB induction was measured at day 1 pre-treatment and a day 14 oftreatment at trough (prior to treatment administration), 1.5 hours, 3hours, 5 hours, and 8 hours following administration of CPI-444 orCPI-444+atezo.

pCREB induction by NECA is attenuated following 14 days of treatment ofCPI-444 and CPI-444+atezo. This attenuation is clearest with treatmentof sub-saturation NECA (1 uM, and 3 uM) in B cells. The 14 day timepoints show the maximal inhibition at 1.5 hr 3 hr and the minimalinhibition at trough and thus reveal the degree of inhibition acrosstime (e.g. whether maximal inhibition is maintained over time).

Example 10

Biomarkers were analyzed in archival tumor tissue and serial biopsies aswell as in peripheral blood to determine whether CPI-444 affectsperipheral and intra-tumoral immune activation and T cell repertoiresand to identify markers that are associated with efficacy.

Example 11

On day 1, prior to drug therapy, blood was collected and stimulated withNECA to induce CREB phosphorylation (pCREB) (FIGS. 24 and 25) and thelevel of uninhibited signaling in B cells and T cells was determined(FIGS. 26-28). On day 14, blood was collected prior to dosing (0 hr,trough) and for a time course post-dose (1.5, 3, 5.5, 8 hr) (FIGS.26-28). The level of signaling was determined and the percent inhibitionon Day 14 compared to Day 1 pre-treatment was calculated.

The relative amount of pCREB inhibition was assessed for each dosingcohort. The majority of patients in the 100 mg BID cohort had thehighest pCREB inhibition at trough and near complete inhibition aftertaking their morning dose (FIG. 34B). Little fluctuation from trough topeak in the 100 mg BID dosing group was observed, demonstrating thatpeak inhibition (at the 3 hr time of peak plasma drug levels) ismaintained through drug trough and therefore 100 mg BID is anappropriate dose for continuous functional inhibition (FIG. 34D). The 50mg BID is not high enough for sustained inhibition (FIG. 34A), and the200 mg QD dose achieves high peak levels but is not maintained at troughas CPI-444 is administered only once per day (FIG. 34C).

Assessing pharmacokinetics and pharmacodynamics revealed a relationshipbetween pCREB percent inhibition and plasma levels of CPI-444. ForCPI-444 plasma levels greater than 2,000 ng/mL, near complete inhibitionof pCREB was observed in both B cells (FIG. 35A) and T cells (FIG. 35B).

Applicants observed a strong correlation between inhibition of pCREB inB cells and inhibition of pCREB in CD4+ T cells (FIG. 36). In thisassay, the pCREB signal is stronger in B cells than CD4+ T cells, thusthe signal-to-noise is better in B cells. This shows that at thepopulation level, pCREB inhibition in B cells is an appropriatesurrogate for directly measuring pCREB in T cells, a cell type ofinterest for CPI-444 activity.

REFERENCES

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Embodiments I Embodiment 1

A method of treating cancer in a subject in need thereof, said methodcomprising administering to said subject a therapeutically effectiveamount of an adenosine-A2A (A2A) receptor antagonist and a programmedcell death protein 1 (PD-1) signaling pathway inhibitor.

Embodiment 2

The method of embodiment 1, wherein said A2A receptor antagonist is acompound of formula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 3

The method of embodiment 2, wherein said A2A receptor antagonist is acompound of formula:

wherein

-   -   R⁶, R^(6.1) and R^(6.2) are independently hydrogen, halogen, ═O,        ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl,        —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,        substituted or unsubstituted alkyl, substituted or unsubstituted        heteroalkyl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocycloalkyl, substituted or        unsubstituted aryl, or substituted or unsubstituted heteroaryl.

Embodiment 4

The method of one of embodiments 1-3, wherein said A2A receptorantagonist is a compound of formula:

Embodiment 5

The method of one of embodiments 1-4, wherein said PD-1 signalingpathway inhibitor is a programmed death-ligand 1 (PD-L1) antagonist or aPD-1 antagonist.

Embodiment 6

The method of embodiment 5, wherein said programmed death-ligand 1(PD-L1) antagonist is an antibody or a small molecule.

Embodiment 7

The method of embodiment 6, wherein said PD-L1 antagonist is anantibody.

Embodiment 8

The method of embodiment 7, wherein said antibody is atezolizumab.

Embodiment 9

The method of embodiment 5, wherein said PD-1 antagonist is an antibodyor a small molecule.

Embodiment 10

The method of any one of embodiments 1-9, wherein said A2A receptorantagonist and said PD-1 signaling pathway inhibitor are administered ina combined synergistic amount.

Embodiment 11

The method of one of embodiments 1-10, wherein said A2A receptorantagonist and said PD-1 signaling pathway inhibitor are administeredsimultaneously or sequentially.

Embodiment 12

The method of one of embodiments 1-11, wherein said A2A receptorantagonist is administered at a first time point and said PD-1 signalingpathway inhibitor is administered at a second time point, wherein saidfirst time point precedes said second time point.

Embodiment 13

The method of embodiment 12, wherein said second time point is withinless than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13,12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2 or 1 days from said first time point.

Embodiment 14

The method of embodiment 12 or 13, wherein said second time point iswithin about 8, 10 or 12 days from said first time point.

Embodiment 15

The method of one of embodiments 1-11, wherein said PD-1 signalingpathway inhibitor is administered at a first time point and said A2Areceptor antagonist is administered at a second time point, wherein saidfirst time point precedes said second time point.

Embodiment 16

The method of embodiment 15, wherein said second time point is withinless than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13,12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2 or 1 days from said first time point.

Embodiment 17

The method of embodiment 15 or 16, wherein said second time point iswithin about 8, 10 or 12 days from said first time point.

Embodiment 18

The method of one of embodiments 1-17, wherein said A2A receptorantagonist is administered at an amount of about 0.5 mg/kg, 1 mg/kg, 5mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg or 300 mg/kg.

Embodiment 19

The method of one of embodiments 1-18, wherein said A2A receptorantagonist is administered at an amount of about 1 mg/kg.

Embodiment 20

The method of one of embodiments 1-18, wherein said PD-1 signalingpathway inhibitor is administered at an amount of less than about 1,300mg.

Embodiment 21

The method of one of embodiments 1-20, wherein said PD-1 signalingpathway inhibitor is administered at an amount of about 1,200 mg.

Embodiment 22

The method of one of embodiments 1-21, wherein said cancer is selectedfrom lung cancer, bladder cancer, melanoma, renal cell carcinoma, coloncancer, ovarian cancer, gastric cancer, breast cancer, head and neckcarcinoma, prostate cancer and a hematologic malignancy.

Embodiment 23

A method of treating cancer in a subject in need thereof, said methodcomprising administering to said subject a therapeutically effectiveamount of an adenosine-A2A (A2A) receptor antagonist, wherein said A2Areceptor antagonist is a compound of formula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 24

The method of embodiment 23, wherein said A2A receptor antagonist is acompound of formula:

wherein

-   -   R⁶, R^(6.1) and R^(6.2) are independently hydrogen, halogen, ═O,        ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl,        —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,        substituted or unsubstituted alkyl, substituted or unsubstituted        heteroalkyl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocycloalkyl, substituted or        unsubstituted aryl, or substituted or unsubstituted heteroaryl.

Embodiment 25

The method of embodiment 23 or 24, wherein said A2A receptor antagonistis a compound of formula:

Embodiment 26

The method of one of embodiments 23-25, further comprising administeringa therapeutically effective amount of a PD-1 signaling pathwayinhibitor.

Embodiment 27

The method of embodiment 26, wherein said A2A receptor antagonist andsaid PD-1 signaling pathway inhibitor are administered in a combinedsynergistic amount.

Embodiment 28

The method of one of embodiments 26-27, wherein said A2A receptorantagonist and said PD-1 signaling pathway inhibitor are administeredsimultaneously or sequentially.

Embodiment 29

The method of one of embodiments 26-28, wherein said A2A receptorantagonist is administered at a first time point and said PD-1 signalingpathway inhibitor is administered at a second time point, wherein saidfirst time point precedes said second time point.

Embodiment 30

The method of embodiment 29, wherein said second time point is withinless than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13,12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days from said first timepoint.

Embodiment 31

The method of embodiment 29 or 30, wherein said second time point iswithin about 8, 10 or 12 days from said first time point.

Embodiment 32

The method of one of embodiments 26-28, wherein said PD-1 signalingpathway inhibitor is administered at a first time point and said A2Areceptor antagonist is administered at a second time point, wherein saidfirst time point precedes said second time point.

Embodiment 33

The method of embodiment 32, wherein said second time point is withinless than about 120, 90, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13,12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days from said first timepoint.

Embodiment 34

The method of embodiment 32 or 33, wherein said second time point iswithin about 8, 10 or 12 days from said first time point.

Embodiment 35

The method of one of embodiments 23-34, wherein said A2A receptorantagonist is administered at an amount of about 0.5 mg/kg, 1 mg/kg, 5mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 200 mg/kg or 300 mg/kg.

Embodiment 36

The method of one of embodiments 23-35, wherein said A2A receptorantagonist is administered at an amount of about 1 mg/kg.

Embodiment 37

The method of one of embodiments 23-35, wherein said PD-1 signalingpathway inhibitor is administered at an amount of less than about 1,300mg.

Embodiment 38

The method of one of embodiments 23-37, wherein said PD-1 signalingpathway inhibitor is administered at an amount of less than about 1,200mg.

Embodiment 39

The method of one of embodiments 23-38, wherein said cancer is selectedfrom lung cancer, bladder cancer, melanoma, renal cell carcinoma, coloncancer, ovarian cancer, gastric cancer, breast cancer, head and neckcarcinoma, prostate cancer and a hematologic malignancy.

Embodiment 40

A method of activating a T cell, said method comprising contacting saidT cell with an A2A receptor antagonist, wherein said A2A receptorantagonist is a compound of formula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 41

The method of embodiment 40, further comprising contacting said T cellwith a PD-1 signaling pathway inhibitor.

Embodiment 42

The method of embodiment 41, wherein said PD-1 signaling pathwayinhibitor is an antibody or a small molecule.

Embodiment 43

The method of one of embodiments 40-42, wherein said T cell is aneffector T cell or a natural killer cell.

Embodiment 44

The method of one of embodiments 40-43, wherein said T cell is anadenosine-suppressed T cell.

Embodiment 45

The method of one of embodiments 40-44, wherein said T cell is a CD8 Tcell.

Embodiment 46

The method of embodiment 45, wherein said CD8 T cell is aCD45RA-negative CD8 Tcell.

Embodiment 47

The method of one of embodiments 40-42, wherein said T cell is a CD4 Tcell.

Embodiment 48

The method of embodiment 47, wherein said CD4 T cell is aCD45RA-negative CD4 Tcell.

Embodiment 49

The method of one of embodiments 40-48, wherein said T cell is within asubject.

Embodiment 50

The method of embodiment 49, wherein said subject is a cancer subject.

Embodiment 51

The method of embodiment 50, wherein said cancer subject is an anti-PD-1refractory subject.

Embodiment 52

A method of inhibiting A2A receptor activity of a cell, said methodcomprising contacting said cell with an A2A receptor antagonist, whereinsaid A2A receptor antagonist is a compound of formula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 53

The method of embodiment 52, wherein said A2A receptor antagonist is acompound of formula:

Embodiment 54

The method of embodiment 52 or 53, wherein said contacting comprisesbinding said A2A receptor antagonist to an A2A receptor of said cell.

Embodiment 55

The method of any one of embodiments 52-54, wherein said cell is a Tcell.

Embodiment 56

The method of embodiment 55, wherein said T cell is an effector T cellor a natural killer cell.

Embodiment 57

The method of embodiment 55, wherein said T cell is a CD8 T cell.

Embodiment 58

The method of embodiment 57, wherein said CD8 T cell is aCD45RA-negative CD8 Tcell.

Embodiment 59

The method of embodiment 55, wherein said T cell is a CD4 Tcell.

Embodiment 60

The method of embodiment 59, wherein said CD4 T cell is aCD45RA-negative CD4 Tcell.

Embodiment 61

The method of one of embodiments 55-60, wherein said T cell is within asubject.

Embodiment 62

The method of embodiment 61, wherein said subject is a cancer subject.

Embodiment 63

The method of embodiment 62, wherein said cancer subject is an anti-PD-1refractory subject.

Embodiment 64

A method of increasing an anti-tumor immune response in a subject inneed thereof, said method comprising administering to said subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist and a programmed cell death protein 1 (PD-1) signalingpathway inhibitor.

Embodiment 65

A method of increasing an anti-tumor immune response in a subject inneed thereof, said method comprising administering to said subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein said A2A receptor antagonist is a compound offormula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 66

The method of embodiment 65, wherein said A2A receptor antagonist is acompound of formula:

Embodiment 67

The method of embodiment 65 or 66, further comprising administering atherapeutically effective amount of a PD-1 signaling pathway inhibitor.

Embodiment 68

The method of embodiment 67, wherein said PD-1 signaling pathwayinhibitor is a PD-L1 antagonist.

Embodiment 69

The method of embodiment 68, wherein said PD-L1 antagonist is a smallmolecule or an antibody.

Embodiment 70

A method of increasing the amount of CD8-positive cells relative to theamount of regulatory T cells in a subject in need thereof, said methodcomprising administering to said subject a therapeutically effectiveamount of an adenosine-A2A (A2A) receptor antagonist and a programmedcell death protein 1 (PD-1) signaling pathway inhibitor.

Embodiment 71

A method of increasing the amount of CD8-positive cells relative to theamount of regulatory T cells in a subject in need thereof, said methodcomprising administering to said subject a therapeutically effectiveamount of an adenosine-A2A (A2A) receptor antagonist, wherein said A2Areceptor antagonist is a compound of formula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 72

The method of embodiment 71, wherein said A2A receptor antagonist is acompound of formula:

Embodiment 73

The method of embodiment 71 or 72, further comprising administering atherapeutically effective amount of a PD-1 signaling pathway inhibitor.

Embodiment 74

The method of embodiment 73, wherein said PD-1 signaling pathwayinhibitor is a PD-L1 antagonist.

Embodiment 75

The method of embodiment 74, wherein said PD-L1 antagonist is a smallmolecule or an antibody.

Embodiment 76

A method of decreasing tumor volume in a subject in need thereof, saidmethod comprising administering to said subject a therapeuticallyeffective amount of an adenosine-A2A (A2A) receptor antagonist and aprogrammed cell death protein 1 (PD-1) signaling pathway inhibitor.

Embodiment 77

A method of decreasing tumor volume in a subject in need thereof, saidmethod comprising administering to said subject a therapeuticallyeffective amount of an adenosine-A2A (A2A) receptor antagonist, whereinsaid A2A receptor antagonist is a compound of formula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 78

The method of embodiment 77, wherein said A2A receptor antagonist is acompound of formula:

Embodiment 79

The method of embodiment 77 or 78, further comprising administering atherapeutically effective amount of a PD-1 signaling pathway inhibitor.

Embodiment 80

The method of embodiment 79, wherein said PD-1 signaling pathwayinhibitor is a PD-L1 antagonist.

Embodiment 81

The method of embodiment 80, wherein said PD-L1 antagonist is a smallmolecule or an antibody.

Embodiment 82

A method of enhancing anti-tumor immune memory in a subject in needthereof, said method comprising administering to said subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist and a programmed cell death protein 1 (PD-1) signalingpathway inhibitor.

Embodiment 83

A method of enhancing anti-tumor immune memory in a subject in needthereof, said method comprising administering to said subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein said A2A receptor antagonist is a compound offormula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁ m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 84

The method of embodiment 83, wherein said A2A receptor antagonist is acompound of formula:

Embodiment 85

The method of embodiment 83 or 84, further comprising administering atherapeutically effective amount of a PD-1 signaling pathway inhibitor.

Embodiment 86

The method of embodiment 85, wherein said PD-1 signaling pathwayinhibitor is a PD-L1 antagonist.

Embodiment 87

The method of embodiment 86, wherein said PD-L1 antagonist is a smallmolecule or an antibody.

Embodiment 88

A method of increasing global immune activation in a subject in needthereof, said method comprising administering to said subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein said A2A receptor antagonist is a compound offormula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 89

The method of embodiment 88, wherein said A2A receptor antagonist is acompound of formula:

Embodiment 90

The method of embodiment 88 or 89, further comprising administering atherapeutically effective amount of a PD-1 signaling pathway inhibitor.

Embodiment 91

The method of embodiment 90, wherein said PD-1 signaling pathwayinhibitor is a PD-L1 antagonist.

Embodiment 92

The method of embodiment 91, wherein said PD-L1 antagonist is a smallmolecule or an antibody.

Embodiment 93

The method of one of embodiments 88-92, wherein said method comprisesactivating a CD4 T cell in said subject.

Embodiment 94

The method of embodiment 93, wherein said CD4 T cell is a memory T cell.

Embodiment 95

The method of embodiment 93, wherein said CD4 T cell is an effector Tcell.

Embodiment 96

The method of one of embodiments 88-95, wherein the relative amount ofCD45RA-negative CD4 T cells in said subject is increased.

Embodiment 97

The method of one of embodiments 88-95, wherein the relative amount ofCD4 T cells in said subject is increased.

Embodiment 98

The method of one of embodiments 88-95, wherein the relative amount ofmemory T cells in said subject is increased.

Embodiment 99

The method of one of embodiments 88-95, wherein the relative amount ofeffector T cells in said subject is increased.

Embodiment 100

The method of one of embodiments 88-95, wherein said method comprisesincreasing the number of PD-1 positive cells in said subject.

Embodiment 101

The method of one of embodiments 88-92, wherein said method comprisesactivating a CD8 T cell in said subject.

Embodiment 102

The method of embodiment 101, wherein the relative amount of CD8 T cellsin said subject is increased.

Embodiment 103

The method of one of embodiments 88-102, wherein the relative frequencyof TCR recombination is increased.

Embodiment 104

The methods of one of embodiments 1, 23, 64, 65, 70, 71, 76, 77, 82, 83or 88 wherein said subject is an anti-PD-1 refractory subject.

Embodiment 105

The method of one of embodiments 1, 26, 64, 67, 76 or 79, wherein saidA2A receptor antagonist is administered at an amount of about 100 mgBID.

Embodiment 106

The method of embodiment 105, wherein said A2A receptor antagonist isadministered for 28 consecutive days.

Embodiment 107

The method of one of embodiments 1, 26, 64, 67, 76, 79, 105 or 106,wherein said PD-1 signaling pathway inhibitor is administered at anamount of about 840 mg.

Embodiment 108

The method of any one of embodiments 105-107, wherein said A2A receptorantagonist is administered at a first time point and said PD-1 signalingpathway inhibitor is administered at a second time point, wherein saidfirst time point precedes said second time point.

Embodiment 109

The method of embodiment 108, wherein said second time point is withinless than about 120, 90, 60, 50, 40, 30, 28, 20, 19, 18, 17, 16, 15, 14,13, 12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2 or 1 days from said first timepoint.

Embodiment 110

The method of embodiment 108 or 109, wherein said second time point iswithin about 14 or 28 days from said first time point.

Embodiment 111

The method of one of embodiments 1, 23, 64 or 65, said method comprisingactivating a T cell in said subject.

Embodiment 112

The method of one of embodiments 1, 23, 64 or 65, said method comprisinginhibiting A2A receptor activity of a cell in said subject.

Embodiment 113

The method of one of embodiments 1, 23, 64 or 65, said method comprisingincreasing an anti-tumor immune response in a subject.

Embodiment 114

The method of one of embodiments 1, 23, 64 or 65, said method comprisingincreasing the amount of CD8-positive cells relative to the amount ofregulatory T cells in said subject.

Embodiment 115

The method of one of embodiments 1, 23, 64 or 65, said method comprisingenhancing anti-tumor immune memory in said subject.

Embodiment 116

The method of one of embodiments 1, 23, 64 or 65, said method comprisingincreasing global immune activation in said subject.

Embodiment 117

A pharmaceutical composition comprising an A2A receptor antagonist, aPD-1 signaling pathway inhibitor and a pharmaceutically acceptableexcipient.

Embodiment 118

The pharmaceutical composition of embodiment 117, wherein said A2Areceptor antagonist is a compound of formula:

wherein,

-   -   R¹ is independently hydrogen, halogen, —CX^(a) ₃, —CN, —SO₂Cl,        —SO_(n1)R⁹, —SO_(v1)NR⁹R¹⁰, —NHNH₂, —ONR⁹R¹⁰, —NHC═(O)NHNH₂,        —NHC═(O)NR⁹R¹⁰, —N(O)_(m1), —NR⁹R¹⁰, —NH—O—R⁹, —C(O)R⁹,        —C(O)—OR⁹, —C(O)NR⁹R¹⁰, —OR⁹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R² is independently hydrogen, halogen, —CX^(b) ₃, —CN, —SO₂Cl,        —SO_(n2)R¹¹, —SO_(v2)NR¹¹R¹², —NHNH₂, —ONR¹¹R¹², —NHC═(O)NHNH₂,        —NHC═(O)NR¹¹R¹², —N(O)_(m2), —NR¹¹R¹², —NH—O—R¹¹, —C(O)R¹¹,        —C(O)—OR¹¹, —C(O)NR¹¹R¹², —OR¹¹, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R³ is independently hydrogen, halogen, —CX^(c) ₃, —CN, —SO₂Cl,        —SO_(n3)R¹³, —SO_(v3)NR¹³R¹⁴, —NHNH₂, —ONR¹³R¹⁴, —NHC═(O)NHNH₂,        —NHC═(O)NR¹³R¹⁴, —N(O)_(m3), —NR¹³R¹⁴, —NH—O—R¹³, —C(O)R¹³,        —C(O)—OR¹³, —C(O)NR¹³R¹⁴, —OR¹³, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted cycloalkyl, substituted or unsubstituted        heterocycloalkyl, substituted or unsubstituted aryl, or        substituted or unsubstituted heteroaryl;    -   R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently hydrogen,        halogen, ═O, ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, substituted or unsubstituted alkyl, substituted        or unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   X^(a), X^(b) and X^(c) are independently —F, —Cl, —Br, or —I;    -   n₁, n₂ and n₃ are independently an integer from 0 to 4;    -   m₁, m₂ and m₃ are independently an integer from 1 to 2; and    -   v₁, v₂ and v₃ are independently an integer from 1 to 2.

Embodiment 119

The pharmaceutical composition of embodiment 118, wherein said A2Areceptor antagonist is a compound of formula:

wherein

-   -   R⁶, R^(6.1) and R^(6.2) are independently hydrogen, halogen, ═O,        ═S, —CF₃, —CN, —CCl₃, —COOH, —CH₂COOH, —CONH₂, —OH, —SH, —SO₂Cl,        —SO₃H, —SO₄H, —SO₂NH₂, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,        substituted or unsubstituted alkyl, substituted or unsubstituted        heteroalkyl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocycloalkyl, substituted or        unsubstituted aryl, or substituted or unsubstituted heteroaryl.

Embodiment 120

The pharmaceutical composition of one of embodiments 117-119, whereinsaid A2A receptor antagonist is a compound of formula:

Embodiment 121

The pharmaceutical composition of one of embodiments 117-120, whereinsaid PD-1 signaling pathway inhibitor is a programmed death-ligand 1(PD-L1) antagonist or a PD-1 antagonist.

Embodiment 122

The pharmaceutical composition of embodiment 121, wherein saidprogrammed death-ligand 1 (PD-L1) antagonist is an antibody or a smallmolecule.

Embodiment 123

The pharmaceutical composition of embodiment 121, wherein said PD-L1antagonist is an antibody.

Embodiment 124

The pharmaceutical composition of embodiment 122 or 123, wherein saidantibody is atezolizumab.

Embodiment 125

The pharmaceutical composition of one of embodiments 121-124, whereinsaid PD-1 antagonist is an antibody or a small molecule.

Embodiment 126

The pharmaceutical composition of one of embodiments 117-125, whereinsaid A2A receptor antagonist and said PD-1 signaling pathway inhibitorare present in a combined synergistic amount, wherein said combinedsynergistic amount is effective to treat cancer in a subject in needthereof.

Embodiments II Embodiment 1

A method of detecting a phosphorylated cAMP response element-bindingprotein (pCREB) in a B-cell or T-cell of a mammalian subject, saidmethod comprising:

-   -   (i) obtaining a blood sample from a mammalian subject;    -   (ii) contacting said blood sample with an adenosine receptor        agonist;    -   (iii) contacting said blood sample with a pCREB detection agent        and a blood cell detection agent, wherein said blood cell        detection agent comprises a B-cell detection agent or T-cell        detection agent, thereby forming a T-cell-detection agent        complex or a B-cell-detection agent complex; and    -   (iv) detecting said T-cell detection agent complex or said        B-cell detection complex thereby detecting said pCREB in a        T-cell or B-cell.

Embodiment 2

The method of embodiment 1, wherein the adenosine receptor agonistcomprises adenosine, 5′-N-Ethylcarboxamidoadenosine (NECA), or an analogthereof.

Embodiment 3

The method of any of embodiments 1 or 2, wherein the pCREB detectionagent comprises an antibody against pCREB.

Embodiment 4

The method of any of embodiments 1-3, wherein the B cell detection agentcomprises an antibody against CD19 and/or an antibody against CD20.

Embodiment 5

The method of any of embodiments 1-4, wherein the T cell detection agentcomprises an antibody against CD3, CD4 and/or an antibody against CD8.

Embodiment 6

The method of any of embodiments 1-5, further comprising contacting saidblood sample with a fixation agent and cell permeabilizing agent aftercontacting the blood sample with an adenonsine receptor agonist andprior to contacting said blood sample with a pCREB detection agent.

Embodiment 7

The method of any of embodiments 1-6, further comprising contacting saidblood sample with an apoptotic cell detection agent.

Embodiment 8

The method of embodiment 7, wherein the apoptotic cell detection agentcomprises an antibody against cPARP.

Embodiment 9

The method of any of embodiments 1-8, further comprising, prior toobtaining said blood sample, administering to said mammalian subject anadenosine receptor antagonist

Embodiment 10

The method of embodiment 9, wherein said adenosine receptor antagonistcomprises an A2a receptor antagonist or an A2b receptor antagonist.

Embodiment 11

The method of embodiments 1-10, further comprising, prior to obtainingsaid blood sample, administering to said mammalian subject ananti-cancer agent.

Embodiment 12

The method of embodiment 11, wherein said anti-cancer agent comprises aPD-L1 antagonist.

Embodiment 13

The method of embodiment 12, wherein said PD-L1 antagonist comprisesatezolizumab.

Embodiment 14

The method of any of embodiments 1-13, further comprising contactingsaid blood sample with a cell subset detection agent.

Embodiment 15

The method of embodiment 14, wherein the cell subset detection agentcomprises a naïve cell detection agent, a memory cell detection agent,or an effector cell detection agent.

Embodiment 16

The method of embodiment 14, wherein said cell subset detection agentcomprises an antibody against CD27 or an antibody against CD45RA.

Embodiment 17

The method of any one of embodiments 1-16, wherein said blood sample iscollected from circulating blood.

Embodiment 18

The method of any one of embodiments 1-16, wherein said blood samplecomprises an intratumoral sample.

Embodiment 19

A method of treating a subject with cancer, said method comprising:

-   -   (i) obtaining a blood sample from a subject with cancer;    -   (ii) detecting a level of pCREB induced by an adenosine receptor        agonist in said sample;    -   (iii) administering an effective amount of an adenosine receptor        antagonist to said subject.

Embodiment 20

The method of embodiment 19, wherein said detecting of said level ofpCREB induced in said sample comprises:

-   -   (a) contacting said blood sample with an adenosine receptor        agonist; and    -   (b) contacting said blood sample with a pCREB detection agent        and a blood cell detection agent, wherein said blood cell        detection agent comprises a B-cell detection agent or T-cell        detection agent.

Embodiment 21

The method of embodiment 20, wherein the pCREB detection agent comprisesan antibody against pCREB.

Embodiment 22

The method of embodiment 20, wherein the B cell detection agentcomprises an antibody against CD19 and/or against CD20.

Embodiment 23

The method of embodiment 20, wherein the T cell detection agentcomprises an antibody against CD3, CD4 and/or an antibody against CD8.

Embodiment 24

The method of embodiments 20-23, wherein said detecting said level ofpCREB induced in said subject comprises measuring a level of pCREB in Bcells or T cells prior to said administering of the effective amount ofan adenosine receptor antagonist to said subject.

Embodiment 25

The method of embodiment 24, further comprising:

-   -   (iv) detecting a level of pCREB induced in said sample following        said administering of the effective amount of adenosine receptor        antagonist to said subject.

Embodiment 26

The method of embodiment 25, wherein said detecting of the level ofpCREB induce in said sample comprises measuring a level of pCREB inducedin B cells or T cells following said administering of the effectiveamount of adenosine receptor antagonist to said subject.

Embodiment 27

The method of embodiment 26, further comprising increasing a dose of anadenosine receptor antagonist based on the level of pCREB induced insaid B cells.

Embodiment 28

A permeabilized blood cell comprising a pCREB detection agent and ablood cell detection agent, wherein said blood cell detection agentcomprises a B-cell detection agent or T-cell detection agent and saidpermeabilized blood cell comprises a permeabilized B-cell orpermeabilized T-cell.

Embodiment 29

The permeabilized blood cell of embodiment 28, further comprising anapoptotic cell detection agent.

Embodiment 30

The permeabilized blood cell of embodiment 29, wherein said apoptoticcell detection agent comprises an antibody against cPARP.

Embodiment 31

The permeabilized blood cell of embodiment 28, further comprising amature cell detection agent.

Embodiment 32

The permeabilized blood cell embodiment 31, wherein said mature celldetection agent comprises antibody against CD27 or an antibody againstCD45RA.

Embodiment 33

A container comprising an adenosine receptor agonist in combination withthe permeabilized cell of embodiment 28.

Embodiment 34

A flow cytometer comprising the permeabilized blood cell of embodiment28.

Embodiments III Embodiment 1

A method of treating cancer in a subject in need thereof, said methodcomprising administering to said subject a therapeutically effectiveamount of an adenosine-A2A (A2A) receptor antagonist of formula:

and a therapeutically effective amount of atezolizumab.

Embodiment 2

The method of embodiment 1, wherein said A2A receptor antagonist andsaid atezolizumab are administered in a combined synergistic amount.

Embodiment 3

The method of embodiment 1 or 2, wherein said adenosine-A2A (A2A)receptor antagonist is administered at 100 mg.

Embodiment 4

The method of one of embodiments 1-3, wherein said adenosine-A2A (A2A)receptor antagonist is administered twice a day (BID).

Embodiment 5

The method of one of embodiments 1-4, wherein said atezolizumab isadministered at 840 mg.

Embodiment 6

The method of one of embodiments 1-5, wherein said atezolizumab isadministered once every two weeks (Q2W).

Embodiment 7

The method of one of embodiments 1-4 or 6, wherein said atezolizumab isadministered at 1200 mg.

Embodiment 8

The method of one of embodiments 1-7, wherein said atezolizumab isadministered once every three weeks (Q3W).

Embodiment 9

The method of one of embodiments 1-8, wherein said cancer is coloncancer, lung cancer, triple negative breast cancer, melanoma, head andneck cancer, prostate cancer, bladder cancer or renal cancer.

Embodiment 10

The method of one of embodiments 1-9, wherein said cancer is coloncancer.

Embodiment 11

The method of one of embodiments 1-9, wherein said cancer is lungcancer.

Embodiment 12

A pharmaceutical composition comprising an adenosine-A2A (A2A) receptorantagonist of formula:

and a pharmaceutically acceptable excipient, wherein said adenosine-A2A(A2A) receptor antagonist is present at 100 mg.

Embodiment 13

The pharmaceutical composition of embodiment 12, further comprisingatezolizumab.

Embodiment 14

The pharmaceutical composition of embodiment 13, wherein said A2Areceptor antagonist and said atezolizumab are present at a combinedsynergistic amount.

Embodiment 15

The pharmaceutical composition of one of embodiments 13-14, wherein saidatezolizumab is present at 840 mg.

Embodiment 16

The pharmaceutical composition of one of embodiments 13-14, wherein saidatezolizumab is present at 1200 mg.

Embodiment 17

The pharmaceutical composition of one of embodiments 12-16, wherein saidpharmaceutical composition is an oral dosage form.

Embodiment 18

A pharmaceutical composition comprising an adenosine-A2A (A2A) receptorantagonist of formula:

atezolizumab and a pharmaceutically acceptable excipient.

Embodiment 19

The pharmaceutical composition of embodiment 18, wherein said A2Areceptor antagonist and said atezolizumab are present at a combinedsynergistic amount.

Embodiment 20

The pharmaceutical composition of embodiment 18 or 19, wherein said A2Areceptor antagonist is present at 100 mg.

Embodiment 21

The pharmaceutical composition of any one of embodiments 18-20, whereinsaid atezolizumab is present at 840 mg.

Embodiment 22

The pharmaceutical composition of any one of embodiments 18-20, whereinsaid atezolizumab is present at 1200 mg.

Embodiment 23

The pharmaceutical composition of any one of embodiments 18-22, whereinsaid pharmaceutical composition is an oral dosage form.

Embodiment 24

A method of treating cancer in a subject in need thereof, said methodcomprising administering to said subject a therapeutically effectiveamount of an adenosine-A2A (A2A) receptor antagonist of formula:

wherein said adenosine-A2A (A2A) receptor antagonist is administered at100 mg twice a day (BID).

Embodiment 25

The method of embodiment 24, wherein said cancer is colon cancer, lungcancer, triple negative breast cancer, melanoma, head and neck cancer,prostate cancer, bladder cancer or renal cancer.

Embodiment 26

The method of embodiment 24 or 25, wherein said cancer is colon cancer.

Embodiment 27

The method of embodiment 24 or 25, wherein said cancer is lung cancer.

Embodiment 28

The method of one of embodiments 24-27, wherein said method furthercomprises administering to said subject a therapeutically effectiveamount of atezolizumab.

Embodiment 29

The method of embodiment 28, wherein said A2A receptor antagonist andsaid atezolizumab are administered in a combined synergistic amount.

Embodiment 30

The method of embodiment 28 or 29, wherein said atezolizumab isadministered at 840 mg.

Embodiment 31

The method of one of embodiments 28-30, wherein said atezolizumab isadministered once every two weeks (Q2W).

Embodiment 32

The method of embodiment 28 or 31, wherein said atezolizumab isadministered at 1200 mg.

Embodiment 33

The method of one of embodiments 28-32, wherein said atezolizumab isadministered once every three weeks (Q3W).

Embodiment 34

A method of activating a T cell, said method comprising contacting saidT cell with an A2A receptor antagonist, wherein said adenosine-A2A (A2A)receptor antagonist is a compound of formula:

Embodiment 35

The method of embodiment 34, wherein said T cell is an effector T cellor a natural killer cell.

Embodiment 36

The method of embodiment 34 or 35, wherein said T cell is anadenosine-suppressed T cell.

Embodiment 37

The method of embodiment 34 or 35, wherein said T cell is a CD8 T cell.

Embodiment 38

The method of embodiment 37, wherein said CD8 T cell is aCD45RA-negative CD8 Tcell.

Embodiment 39

The method of any one of embodiments 34-38, wherein said T cell iswithin a subject.

Embodiment 40

A method of inhibiting A2A receptor activity of a cell, said methodcomprising contacting said cell with an A2A receptor antagonist, whereinsaid A2A receptor antagonist is a compound of formula:

Embodiment 41

The method of embodiment 40, wherein said contacting comprises bindingsaid A2A receptor antagonist to an A2A receptor of said cell.

Embodiment 42

The method of any one of embodiments 40-41, wherein said cell is a Tcell.

Embodiment 43

The method of embodiment 42, wherein said T cell is an effector T cellor a natural killer cell.

Embodiment 44

The method of embodiment 42, wherein said T cell is a CD45RA-negativeCD8 Tcell.

Embodiment 45

The method of one of embodiments 42-44, wherein said T cell is within asubject.

Embodiment 46

The method of embodiment 45, wherein said subject is a cancer subject.

Embodiment 47

The method of embodiment 46, wherein said cancer subject is an anti-PD-1refractory subject.

Embodiment 48

A method of increasing an anti-tumor immune response in a subject inneed thereof, said method comprising administering to said subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein said adenosine-A2A (A2A) receptor antagonist is acompound of formula:

Embodiment 49

The method of embodiment 48, further comprising administering atherapeutically effective amount of atezolizumab.

Embodiment 50

The method of embodiment 48 or 49, wherein said adenosine-A2A (A2A)receptor antagonist is administered at 100 mg twice a day (BID).

Embodiment 51

The method of any one of embodiments 48-50, wherein said atezolizumab isadministered at 840 mg once every two weeks (Q2W).

Embodiment 52

The method of any one of embodiments 48-50, wherein said atezolizumab isadministered at 1200 mg once every three weeks (Q3W).

Embodiment 53

A method of increasing the amount of CD8-positive cells relative to theamount of regulatory T cells in a subject in need thereof, said methodcomprising administering to said subject a therapeutically effectiveamount of an adenosine-A2A (A2A) receptor antagonist, wherein saidadenosine-A2A (A2A) receptor antagonist is a compound of formula:

Embodiment 54

The method of embodiment 53, further comprising administering atherapeutically effective amount of atezolizumab.

Embodiment 55

The method of embodiment 53 or 54, wherein said adenosine-A2A (A2A)receptor antagonist is administered at 100 mg twice a day (BID).

Embodiment 56

The method of any one of embodiments 53-55, wherein said atezolizumab isadministered at 840 mg once every two weeks (Q2W).

Embodiment 57

The method of any one of embodiments 53-55, wherein said atezolizumab isadministered at 1200 mg once every three weeks (Q3W).

Embodiment 58

A method of decreasing tumor volume in a subject in need thereof, saidmethod comprising administering to said subject a therapeuticallyeffective amount of an adenosine-A2A (A2A) receptor antagonist, whereinsaid adenosine-A2A (A2A) receptor antagonist is a compound of formula:

Embodiment 59

The method of embodiment 58, further comprising administering atherapeutically effective amount of atezolizumab.

Embodiment 60

The method of embodiment 58 or 59, wherein said adenosine-A2A (A2A)receptor antagonist is administered at 100 mg twice a day (BID).

Embodiment 61

The method of any one of embodiments 58-60, wherein said atezolizumab isadministered at 840 mg once every two weeks (Q2W).

Embodiment 62

The method of any one of embodiments 58-60, wherein said atezolizumab isadministered at 1200 mg once every three weeks (Q3W).

Embodiment 63

A method of enhancing anti-tumor immune memory in a subject in needthereof, said method comprising administering to said subject atherapeutically effective amount of an adenosine-A2A (A2A) receptorantagonist, wherein said adenosine-A2A (A2A) receptor antagonist is acompound of formula:

Embodiment 64

The method of embodiment 63, further comprising administering atherapeutically effective amount of atezolizumab.

Embodiment 65

The method of embodiment 63 or 64, wherein said adenosine-A2A (A2A)receptor antagonist is administered at 100 mg twice a day (BID).

Embodiment 66

The method of any one of embodiments 63-65, wherein said atezolizumab isadministered at 840 mg once every two weeks (Q2W).

Embodiment 67

The method of any one of embodiments 63-65, wherein said atezolizumab isadministered at 1200 mg once every three weeks (Q3W).

What is claimed is:
 1. A method of treating cancer in a subject in needthereof, said method comprising administering to said subject atherapeutically effective amount of an adenosine-A2A receptor antagonistof formula:

and a therapeutically effective amount of atezolizumab; wherein thecancer is lymphoma, colon cancer, non-small cell lung cancer, triplenegative breast cancer, melanoma, head and neck cancer, prostate cancer,bladder cancer, colorectal cancer, or renal cancer.
 2. The method ofclaim 1, wherein said adenosine-A2A receptor antagonist and saidatezolizumab are administered in a combined synergistic amount.
 3. Themethod of claim 1, wherein said adenosine-A2A receptor antagonist isadministered at 100 mg.
 4. The method of claim 1, wherein saidadenosine-A2A receptor antagonist is administered twice a day.
 5. Themethod of claim 1, wherein said atezolizumab is administered at 840 mg.6. The method of claim 1, wherein said atezolizumab is administered onceevery two weeks.
 7. The method of claim 1, wherein said atezolizumab isadministered at 1200 mg.
 8. The method of claim 1, wherein saidatezolizumab is administered once every three weeks.
 9. The method ofclaim 1, wherein said cancer is colon cancer, non-small cell lungcancer, triple negative breast cancer, melanoma, prostate cancer, orrenal cancer.
 10. The method of claim 1, wherein the cancer is renalcancer.
 11. The method of claim 1, wherein the cancer is renal cancer ornon-small cell lung cancer.
 12. The method of claim 1, wherein thecancer is renal cancer, non-small cell lung cancer, or prostate cancer.